Enzyme method

ABSTRACT

The invention relates to a new method of characterizing a target polynucleotide. The method uses a pore and a Hel308 helicase or amolecular motor which is capable of binding to the target polynucleotide at an internal nucleotide. The helicase or molecular motor controls the movement of the target polynucleotide through the pore.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15,674,653, filed Aug. 11, 2017, which is a continuation of U.S. patentapplication Ser. No. 14/351,038, filed Apr. 10, 2014, which is anational stage filing under 35 U.S.C. 371 of PCT InternationalApplication No. PCT/GB2012/052579, filed Oct. 18, 2012, which claims thebenefit under 35 U.S.C. § 119(e) to U.S. Provisional Patent ApplicationNo. 61/599,244, filed Feb. 15, 2012 and U.S. Provisional PatentApplication No. 61/549,998, filed Oct. 21, 2011; the entire contents ofeach of which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to a new method of characterising a targetpolynucleotide. The method uses a pore and a Hel308 helicase or amolecular motor which is capable of binding to the target polynucleotideat an internal nucleotide. The helicase or molecular motor controls themovement of the target polynucleotide through the pore.

BACKGROUND OF THE INVENTION

There is currently a need for rapid and cheap polynucleotide (e.g. DNAor RNA) sequencing and identification technologies across a wide rangeof applications. Existing technologies are slow and expensive mainlybecause they rely on amplification techniques to produce large volumesof polynucleotide and require a high quantity of specialist fluorescentchemicals for signal detection.

Transmembrane pores (nanopores) have great potential as direct,electrical biosensors for polymers and a variety of small molecules. Inparticular, recent focus has been given to nanopores as a potential DNAsequencing technology.

When a potential is applied across a nanopore, there is a change in thecurrent flow when an analyte, such as a nucleotide, resides transientlyin the barrel for a certain period of time. Nanopore detection of thenucleotide gives a current change of known signature and duration. Inthe “Strand Sequencing” method, a single polynucleotide strand is passedthrough the pore and the identity of the nucleotides are derived. StrandSequencing can involve the use of a nucleotide handling protein tocontrol the movement of the polynucleotide through the pore.

SUMMARY OF THE INVENTION

The inventors have demonstrated that a Hel308 helicase can control themovement of a polynucleotide through a pore especially when a potential,such as a voltage, is applied. The helicase is capable of moving atarget polynucleotide in a controlled and stepwise fashion against orwith the field resulting from the applied voltage. Surprisingly, thehelicase is capable of functioning at a high salt concentration which isadvantageous for characterising the polynucleotide and, in particular,for determining its sequence using Strand Sequencing. This is discussedin more detail below.

Accordingly, the invention provides a method of characterising a targetpolynucleotide, comprising:

(a) contacting the target polynucleotide with a transmembrane pore and aHel308 helicase such that the helicase controls the movement of thetarget polynucleotide through the pore and nucleotides in the targetpolynucleotide interact with the pore; and

(b) measuring one or more characteristics of the target polynucleotideduring one or more interactions and thereby characterising the targetpolynucleotide.

The Invention Also Provides:

a method of forming a sensor for characterising a target polynucleotide,comprising forming a complex between a pore and a Hel308 helicase andthereby forming a sensor for characterising the target polynucleotide;

-   -   use of a Hel308 helicase to control the movement of a target        polynucleotide through a pore;    -   a kit for characterising a target polynucleotide comprising (a)        a pore and (b) a Hel308 helicase; and    -   an analysis apparatus for characterising target polynucleotides        in a sample, comprising a plurality of pores and a plurality of        a Hel308 helicase.

The inventors have also demonstrated that a molecular motor which iscapable of binding to a target polynucleotide at an internal nucleotidecan control the movement of the polynucleotide through a pore especiallywhen a potential, such as a voltage, is applied. The motor is capable ofmoving the target polynucleotide in a controlled and stepwise fashionagainst or with the field resulting from the applied voltage.Surprisingly, when the motor is used in the method of the invention itis possible to control the movement of an entire strand of targetpolynucleotide through a nanopore. This is advantageous forcharacterising the polynucleotide and, in particular, for determiningits sequence using Strand Sequencing.

Hence, the invention also provides a method of characterising a targetpolynucleotide, comprising:

(a) contacting the target polynucleotide with a transmembrane pore and amolecular motor which is capable of binding to the target polynucleotideat an internal nucleotide such that the molecular motor controls themovement of the target polynucleotide through the pore and nucleotidesin the target polynucleotide interact with the pore; and

(b) measuring one or more characteristics of the target polynucleotideduring one or more interactions and thereby characterising the targetpolynucleotide.

DESCRIPTION OF THE FIGURES

FIG. 1A. Example schematic of use of a helicase to control DNA movementthrough a nanopore. 1) A ssDNA substrate with an annealed primercontaining a cholesterol-tag is added to the cis side of the bilayer.The cholesterol tag binds to the bilayer, enriching the substrate at thebilayer surface. 2) Helicase added to the cis compartment binds to theDNA. In the presence of divalent metal ions and NTP substrate, thehelicase moves along the DNA. 3) Under an applied voltage, the DNAsubstrate is captured by the nanopore via the leader section on the DNA.The DNA is pulled through the pore under the force of the appliedpotential until a helicase, bound to the DNA, contacts the top of thepore, preventing further uncontrolled DNA translocation. During thisprocess dsDNA sections (such as the primer) are removed. The helicasemovement along the DNA in a 3′ to 5′ direction pulls the threaded DNAout of the pore against the applied field. 4) The helicase pulls the DNAout of the nanopore, feeding it back to the cis compartment. The lastsection of DNA to pass through the nanopore is the 5′-leader. 5) Whenthe helicase moves the DNA out of the nanopore it is lost back to thecis compartment.

FIG. 1B. A DNA substrate design used in the Example.

FIG. 2 . Helicase is able to move DNA through a nanopore in a controlledfashion, producing stepwise changes in current as the DNA moves throughthe nanopore. Example 25 helicase-DNA events (180 mV, 400 mM KCl, HepespH 8.0, 0.15 nM 400 mer DNA, 100 nM Hel308 Mbu, 1 mM DTT, 1 mM ATP, 1 mMMgCl₂). Top) Section of current vs. time acquisition of Hel308 400merDNA events. The open-pore current is ˜180 pA. DNA is captured by thenanopore under the force of the applied potential (+180 mV). DNA withenzyme attached results in a long block (at ˜60 pA in this condition)that shows stepwise changes in current as the enzyme moves the DNAthrough the pore. Middle) The middle section is an enlargement of one ofthe DNA events, showing DNA-enzyme capture, stepwise current changes asthe DNA is pulled through the pore, and ending in a characteristic longpolyT level before exiting the nanopore. Bottom) enlargement of thestepwise changes in current as DNA is moved through the nanopore.

FIGS. 3A-3B. Helicase controlled DNA movement resulting in a consistentpattern of current transitions as DNA is passed through the nanopore.Examples of the last ˜80 current transitions from four typical DNAevents that end in the polyT level. The four examples (two in FIG. 3Aand two in FIG. 3B) illustrate that a consistent pattern of currenttransitions are observed.

FIGS. 4A-4D. Increased salt concentration increases pore current andgives a larger DNA discrimination range (range=minimum current tomaximum current across the DNA current transitions). Examplehelicase-DNA events (180 mV, Hepes pH 8.0, 0.15 nM 400mer DNA SEQ IDNOs: 59 and 60, 100 nM Hel308 Mbu, 1 mM DTT, 1 mM ATP, 1 mM MgCl₂) at400 mM, 1 M, and 2 M KCl are shown in FIGS. 4A-4C. Top traces show afull event that ends in the polyT level, and lower traces show a zoomsection of the last 10 seconds of each event with a constant y-axiscurrent scale of 150 pA. Increasing the salt concentration from 400 mMKCl to 2M KCl leads to a ˜350% increase in the open-pore current (I-openfrom ˜180 pA to ˜850 pA), and a ˜200% increase in discrimination range(˜25 pA to ˜75 pA). FIG. 4D is a plot of DNA discrimination range as afunction of salt concentration.

FIGS. 5A-5B. The helicase can control the movement of DNA in at leasttwo modes of operation. The helicase moves along the DNA in the 3′-5′direction, but the orientation of the DNA in the nanopore (dependent onwhich end of the DNA is captured) means that the enzyme can be used toeither move the DNA out of the nanopore against the applied field, ormove the DNA into the nanopore with the applied field. FIG. 5A. When the5′ end of the DNA is captured the helicase works against the directionof the field applied by the voltage, pulling the threaded DNA out of thenanopore until the DNA is ejected back to the cis chamber. On the rightis an example DNA-helicase event from Hel308 running 5′ down against theapplied field. FIG. 5B. When the DNA is captured 3′-down in thenanopore, the enzyme moves the DNA into the nanopore in the direction ofthe field until it is fully translocated through the pore and lost onthe trans side of the bilayer. On the right is an example DNA-helicaseevent from Hel308 running 3′-down with the applied field. Current tracesvary between the 5′ down and 3′ down orientations of DNA.

FIGS. 6A-6B. Fluorescence assay for testing enzyme activity. FIG. 6A. Acustom fluorescent substrate was used to assay the ability of thehelicase to displace hybridised dsDNA. 1) The fluorescent substratestrand (100 nM final) has a 3′ ssDNA overhang, and a 40 base section ofhybridised dsDNA. The major upper strand has a carboxyfluorescein baseat the 5′ end, and the hybridised complement has a black-hole quencher(BHQ-1) base at the 3′ end. When hybridised the fluorescence from thefluorescein is quenched by the local BHQ-1, and the substrate isessentially non-fluorescent. 1 μM of a capture strand that iscomplementary to the shorter strand of the fluorescent substrate isincluded in the assay. 2) In the presence of ATP (1 mM) and MgCl₂ (5mM), helicase (100 nM) added to the substrate binds to the 3′ tail ofthe fluorescent substrate, moves along the major strand, and displacesthe complementary strand as shown. 3) Once the complementary strand withBHQ-1 is fully displaced the fluorescein on the major strand fluoresces.4) Excess of capture strand preferentially anneals to the complementaryDNA to prevent re-annealing of initial substrate and loss offluorescence. FIG. 6B. Graph of the initial rate of activity in buffersolutions (10 mM Hepes pH 8.0, 1 mM ATP, 5 mM MgCl₂, 100 nM fluorescentsubstrate DNA, 1 μM capture DNA) containing different concentrations ofKCl from 400 mM to 2 M.

FIGS. 7A-7C show examples of helicase controlled DNA events usingdifferent Hel308 helicases (180 mV, Hepes pH 8.0, 0.15 nM 400mer DNA SEQID NOs: 59 and 60, 100 nM Hel308, 1 mM DTT, 1 mM ATP, 1 mM MgCl₂):Hel308 Mhu (FIG. 7A), Hel308 Mok (FIG. 7B) and Hel308 Mma (FIG. 7C).These represent typical examples of DNA controlled movement through MspAnanopores that ended at the polyT level.

FIG. 8 . Fluorescence assay for testing helicase internal bindingactivity. Panel A) Custom fluorescent substrates were used to assay theability of the helicases to bind to DNA lacking native 3′ ends, allowingthem to subsequently displace hybridised dsDNA. The fluorescentsubstrate strand (50 nM final) has a 3′ ssDNA overhang, and a 40 basesection of hybridised dsDNA. The major upper strands are modified withfour consecutive non-DNA-derived triethylene glycol spacers (referred toas “spacer 9” groups), either at the 3′ end, or internally, at thejunction between the overhang and the dsDNA (as a negative control).Furthermore, the major upper strand has a carboxyfluorescein base at the5′ end, and the hybridised complement has a black-hole quencher (BHQ-1)base at the 3′ end. When hybridised, the fluorescence from thefluorescein is quenched by the local BHQ-1, and the substrate isessentially non-fluorescent. A capture strand (1 μM), that iscomplementary to the shorter strand of the fluorescent substrate, isincluded in the assay. Panel B) In the presence of ATP (1 mM) and MgCl₂(1 mM), a Hel308 helicase homologue (20 nM), added to the substratecontaining 3′-terminal “spacer 9” groups, can bind to the ssDNA overhangof the fluorescent substrate, move along the major strand, and displacethe complementary strand. Panel C) Once the complementary strand withBHQ-1 is fully displaced the fluorescein on the major strand fluoresces.Panel D) An excess of capture strand preferentially anneals to thecomplementary DNA to prevent re-annealing of initial substrate and lossof fluorescence.

FIG. 9 shows the relative rates of Hel308-mediated dsDNA turnovercomparing 3′-unmodified DNA and 3′-“spacer 9” DNA in 400 mM NaCl, 10 mMHepes, pH 8.0, 1 mM ATP, 1 mM MgCl₂, 50 nM fluorescent substrate DNA, 1μM capture DNA.

FIG. 10 . Schematic of the use of a helicase to control DNA movementthrough a nanopore which is employed in example 5. Panel A) A DNAsubstrate (SEQ ID NOs 67 and 68) with an annealed primer (SEQ ID NO 69)with an attached cholesterol-tag is added to the cis side of thebilayer. The cholesterol tag binds to the bilayer, enriching thesubstrate at the bilayer surface. Helicase added to the cis compartmentbinds to the 4 bp leader of SEQ ID NO 67. Panel B) Under an appliedvoltage, the DNA substrate is captured by the nanopore via the 5′ leadersection on the DNA, which strips off SEQ ID NO 69. Panel C) Under theforce of the applied field the DNA is pulled into the pore until thebound helicase contacts the top of the pore and prevents furtheruncontrolled translocation. In this process the antisense strand SEQ IDNO 68 is stripped from the DNA strand. Panel D) In the presence ofdivalent metal ions and NTP substrate, the helicase on top of the poremoves along the DNA and controls the translocation of the DNA throughthe pore. The helicase movement along the DNA in a 3′ to 5′ directionpulls the threaded DNA out of the pore against the applied field. Theexposed single stranded DNA on the cis side (3′ in this case) isavailable for further helicases to bind either at the terminalnucleotide or at an internal nucleotide. Panel E) If the helicase at thepore disengages from the DNA, the DNA is pulled into the pore by thefield until the next helicase on the DNA reaches the pore. The helicaseat the pore pulls the DNA out of the nanopore, feeding it back to thecis compartment. The last section of DNA to pass through the nanopore isthe 5′-leader. Panel F) When the helicase moves the DNA out of thenanopore it is lost back to the cis compartment. Arrows indicate thedirection of DNA movement.

FIG. 11 shows data plots which indicate how the position of the regionof DNA in the nanopore of the 900 mer (y-axis) varied as the Hel308helicase homologue Mbu controlled the translocation of the DNA strandthrough the MspA pore (x-axis) during each helicase event. Panels A-Cshow examples of typical translocation events of the entire DNA strandfrom approximately the beginning of the strand through to the end of thestrand (exiting via polyT leader), whereas Panel D shows an example ofincomplete DNA translocation, where enzyme dettachment means the DNAnever makes it to the end of the strand. The slips (eg. such as thelarge slips highlighted by dotted circles) indicate the sequence fallingback to a previous point in the strand, and are the result of enzymedettachment. When an enzyme dettaches the DNA will be pulled back underthe force of the field into the nanopore until another enzyme furtheralong the strand contacts the pore, then continuing helicase movement.

FIG. 12 shows data plots which indicate how the position of the 900 mervaried as the Hel308 helicase homologue Tga controlled the translocationof the DNA strand through the MspA pore. Panels A-D show translocationof the entire DNA strand.

FIG. 13 shows a fluorescence assay used to compare the enzymeprocessivity of Hel308 Mbu helicase (SEQ ID NO: 10) to that of Hel 308Mok helicase (SEQ ID NO: 29). A custom fluorescent substrate was used toassay the ability of the helicase to displace hybridised dsDNA. Thefluorescent substrate (50 nM final) has a 3′ ssDNA overhang, and 80 and33 base-pair sections of hybridised dsDNA (Panel A, SEQ ID NO: 70). Themajor bottom “template” strand is hybridised to an 80 nt “blocker”strand (SEQ ID NO: 71), adjacent to its 3′ overhang, and a 33 ntfluorescent probe (SEQ ID NO: 72), labelled at its 5′ and 3′ ends withcarboxyfluorescein (FAM) and black-hole quencher (BHQ-1) bases,respectively. When hybridised, the FAM is distant from the BHQ-1 and thesubstrate is essentially fluorescent. In the presence of ATP (1 mM) andMgCl₂ (10 mM), the helicase (20 nM) binds to the substrate's 3′ overhang(SEQ ID NO: 70), moves along the lower strand, and begins to displacethe 80 nt blocker strand (SEQ ID NO: 71), as shown in Panel B. Ifprocessive, the helicase displaces the fluorescent probe too (Panel C,SEQ ID NO: 72, labeled with a carboxyfluorescein (FAM) at its 5′ end ablack-hole quencher (BHQ-1) at its 3′ end). The fluorescent probe isdesigned in such a way that its 5′ and 3′ ends are self-complementaryand thus form a kinetically-stable hairpin once displaced, preventingthe probe from re-annealing to the template strand (Panel D). Uponformation of the hairpin product, the FAM is brought into the vicinityof the BHQ-1 and its fluorescence is quenched. A processive enzyme,capable of displacing the 80 mer “blocker” (SEQ ID NO: 71) andfluorescent (SEQ ID NO: 72, labeled with a carboxyfluorescein (FAM) atits 5′ end a black-hole quencher (BHQ-1) at its 3′ end) strands willtherefore lead to a decrease in fluorescence over time. However, if theenzyme has a processivity of less than 80 nt it would be unable todisplace the fluorescent strand (SEQ ID NO: 72, labeled with acarboxyfluorescein (FAM) at its 5′ end a black-hole quencher (BHQ-1) atits 3′ end) and, therefore, the “blocker” strand (SEQ ID NO: 71) wouldreanneal to the major bottom strand (Panel E).

FIG. 14 shows additional custom fluorescent substrates which were alsoused for control purposes. The substrate used as a negative control wasidentical to that of the one described in FIGS. 3A-3B but lacking the 3′overhang (Panel A, (SEQ ID NOs: 71, 72 (labeled with acarboxyfluorescein (FAM) at its 5′ end a black-hole quencher (BHQ-1) atits 3′ end) and 73)). A similar substrate to that described in FIGS.3A-3B but lacking the 80 base pair section (SEQ ID NOs: 72 (labeled witha carboxyfluorescein (FAM) at its 5′ end a black-hole quencher (BHQ-1)at its 3′ end) and 74), was used as a positive control for active, butnot necessarily processive, helicases (Panel B).

FIG. 15 shows a graph of the time-dependent fluorescence changes upontesting Hel308 Mbu helicase (SEQ ID NO: 10) and Hel 308 Mok helicase(SEQ ID NO: 29) against the processivity substrate shown in FIG. 13 inbuffered solution (400 mM NaCl, 10 mM Hepes pH 8.0, 1 mM ATP, 10 mMMgCl₂, 50 nM fluorescent substrate DNA (SEQ ID NOs: 70, 71 and 72(labeled with a carboxyfluorescein (FAM) at its 5′ end a black-holequencher (BHQ-1) at its 3′ end). The decrease in fluorescence exhibitedby Hel308 Mok denotes the increased processivity of these complexes ascompared to Hel308 Mbu (SEQ ID NO: 10).

FIG. 16 shows a graph of the time-dependent fluorescence changes upontesting Hel308 Mbu helicase (SEQ ID NO: 10) and Hel 308 Mok helicase(SEQ ID NO: 29) against the positive control processivity substrate(shown in FIG. 14 Panel B, SEQ ID NOs: 72 (labeled with acarboxyfluorescein (FAM) at its 5′ end a black-hole quencher (BHQ-1) atits 3′ end) and 74) in buffered solution (400 mM NaCl, 10 mM Hepes pH8.0, 1 mM ATP, 10 mM MgCl₂, 50 nM fluorescent substrate DNA (SEQ ID NOs:72 (labeled with a carboxyfluorescein (FAM) at its 5′ end a black-holequencher (BHQ-1) at its 3′ end) and 74)). This positive controldemonstrated that both helicases were indeed active, as denoted by afluorescence decrease for both samples.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 shows the codon optimised polynucleotide sequence encodingthe MS-B1 mutant MspA monomer. This mutant lacks the signal sequence andincludes the following mutations: D90N, D91N, D93N, D118R, D134R andE139K.

SEQ ID NO: 2 shows the amino acid sequence of the mature form of theMS-B1 mutant of the MspA monomer. This mutant lacks the signal sequenceand includes the following mutations: D90N, D91N, D93N, D118R, D134R andE139K.

SEQ ID NO: 3 shows the polynucleotide sequence encoding one subunit ofα-hemolysin-E111N/K147N (α-HL-NN; Stoddart et al., PNAS, 2009; 106(19):7702-7707).

SEQ ID NO: 4 shows the amino acid sequence of one subunit of α-HL-NN.

SEQ ID NOs: 5 to 7 shows the amino acid sequences of MspB, C and D.

SEQ ID NO: 8 shows the amino acid sequence of the Hel308 motif.

SEQ ID NO: 9 shows the amino acid sequence of the extended Hel308 motif.

SEQ ID NOs: 10 to 58 show the amino acid sequences of the Hel308helicases and motifs in Table 5.

SEQ ID NOs: 59 to 74 show the sequences used in the Examples.

SEQ ID NO: 75 shows the sequence of Hel308 Dth in the alignment on page57 onwards.

SEQ ID NO: 76 shows the sequence of Hel308 Mmar in the alignment on page57 onwards.

SEQ ID NO: 77 shows the sequence of Hel308 Nth in the alignment on page57 onwards.

SEQ ID NO: 78 shows the consensus sequence in the alignment on page 57onwards.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that different applications of the disclosedproducts and methods may be tailored to the specific needs in the art.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

In addition as used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “apore” includes two or more such pores, reference to “a helicase”includes two or more such helicases, reference to “a polynucleotide”includes two or more such polynucleotides, and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

Hel308 Methods of the Invention

The invention provides a method of characterising a targetpolynucleotide. The method comprises contacting the targetpolynucleotide with a transmembrane pore and a Hel308 helicase such thatthe helicase controls the movement of the target polynucleotide throughthe pore and nucleotides in the target polynucleotide interact with thepore. One or more characteristics of the target polynucleotide are thenmeasured using standard methods known in the art. Steps (a) and (b) arepreferably carried out with a potential applied across the pore. Asdiscussed in more detail below, the applied potential typically resultsin the formation of a complex between the pore and the helicase. Theapplied potential may be a voltage potential. Alternatively, the appliedpotential may be a chemical potential. An example of this is using asalt gradient across the lipid membrane. A salt gradient is disclosed inHolden et al., J Am Chem Soc. 2007 Jul 11; 129(27):8650-5.

In some instances, the current passing through the pore during one ormore interactions is used to determine the sequence of the targetpolynucleotide. This is Strand Sequencing.

The method has several advantages. First, the inventors havesurprisingly shown that Hel308 helicases have a surprisingly high salttolerance and so the method of the invention may be carried out at highsalt concentrations. In the context of Strand Sequencing, a chargecarrier, such as a salt, is necessary to create a conductive solutionfor applying a voltage offset to capture and translocate the targetpolynucleotide and to measure the resulting sequence-dependent currentchanges as the polynucleotide passes through the pore. Since themeasurement signal is dependent on the concentration of the salt, it isadvantageous to use high salt concentrations to increase the magnitudeof the acquired signal. High salt concentrations provide a high signalto noise ratio and allow for currents indicative of the presence of anucleotide to be identified against the background of normal currentfluctuations. For Strand Sequencing, salt concentrations in excess of100 mM are ideal and salt concentrations of 1 M and above are preferred.The inventors have surprisingly shown that Hel308 helicases willfunction effectively at salt concentrations as high as, for example, 2M.

Second, when a voltage is applied, Hel308 helicases can surprisinglymove the target polynucleotide in two directions, namely with or againstthe field resulting from the applied voltage. Hence, the method of theinvention may be carried out in one of two preferred modes. Differentsignals are obtained depending on the direction the targetpolynucleotide moves through the pore, ie in the direction of or againstthe field. This is discussed in more detail below.

Third, Hel308 helicases typically move the target polynucleotide throughthe pore one nucleotide at a time. Hel308 helicases can thereforefunction like a single-base ratchet. This is of course advantageous whensequencing a target polynucleotide because substantially all, if notall, of the nucleotides in the target polynucleotide may be identifiedusing the pore.

Fourth, Hel308 helicases are capable of controlling the movement ofsingle stranded polynucleotides and double stranded polynucleotides.This means that a variety of different target polynucleotides can becharacterised in accordance with the invention.

Fifth, Hel308 helicases appear very resistant to the field resultingfrom applied voltages. The inventors have seen very little movement ofthe polynucleotide under an “unzipping” condition. This is importantbecause it means that there are no complications from unwanted“backwards” movements when moving polynucleotides against the fieldresulting from an applied voltage.

Sixth, Hel308 helicases are easy to produce and easy to handle. Theiruse therefore contributed to a straightforward and less expensive methodof sequencing.

The method of the invention is for characterising a targetpolynucleotide. A polynucleotide, such as a nucleic acid, is amacromolecule comprising two or more nucleotides. The polynucleotide ornucleic acid may comprise any combination of any nucleotides. Thenucleotides can be naturally occurring or artificial. One or morenucleotides in the target polynucleotide can be oxidized or methylated.One or more nucleotides in the target polynucleotide may be damaged. Oneor more nucleotides in the target polynucleotide may be modified, forinstance with a label or a tag. The target polynucleotide may compriseone or more spacers.

A nucleotide typically contains a nucleobase, a sugar and at least onephosphate group. The nucleobase is typically heterocyclic. Nucleobasesinclude, but are not limited to, purines and pyrimidines and morespecifically adenine, guanine, thymine, uracil and cytosine. The sugaris typically a pentose sugar. Nucleotide sugars include, but are notlimited to, ribose and deoxyribose. The nucleotide is typically aribonucleotide or deoxyribonucleotide. The nucleotide typically containsa monophosphate, diphosphate or triphosphate. Phosphates may be attachedon the 5′ or 3′ side of a nucleotide.

Nucleotides include, but are not limited to, adenosine monophosphate(AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP),uridine monophosphate (UMP), cytidine monophosphate (CMP), cyclicadenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP),deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate(dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate(dUMP) and deoxycytidine monophosphate (dCMP). The nucleotides arepreferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP ordCMP.

A nucleotide may be abasic (i.e. lack a nucleobase).

The polynucleotide may be single stranded or double stranded. At least aportion of the polynucleotide is preferably double stranded.

The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid(DNA) or ribonucleic acid (RNA). The target polynucleotide can compriseone strand of RNA hybridized to one strand of DNA. The polynucleotidemay be any synthetic nucleic acid known in the art, such as peptidenucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid(TNA), locked nucleic acid (LNA) or other synthetic polymers withnucleotide side chains.

The whole or only part of the target polynucleotide may be characterisedusing this method. The target polynucleotide can be any length. Forexample, the polynucleotide can be at least 10, at least 50, at least100, at least 150, at least 200, at least 250, at least 300, at least400 or at least 500 nucleotide pairs in length. The polynucleotide canbe 1000 or more nucleotide pairs, 5000 or more nucleotide pairs inlength or 100000 or more nucleotide pairs in length.

The target polynucleotide is present in any suitable sample. Theinvention is typically carried out on a sample that is known to containor suspected to contain the target polynucleotide. Alternatively, theinvention may be carried out on a sample to confirm the identity of oneor more target polynucleotides whose presence in the sample is known orexpected.

The sample may be a biological sample. The invention may be carried outin vitro on a sample obtained from or extracted from any organism ormicroorganism. The organism or microorganism is typically archaean,prokaryotic or eukaryotic and typically belongs to one the fivekingdoms: plantae, animalia, fungi, monera and protista. The inventionmay be carried out in vitro on a sample obtained from or extracted fromany virus. The sample is preferably a fluid sample. The sample typicallycomprises a body fluid of the patient. The sample may be urine, lymph,saliva, mucus or amniotic fluid but is preferably blood, plasma orserum. Typically, the sample is human in origin, but alternatively itmay be from another mammal animal such as from commercially farmedanimals such as horses, cattle, sheep or pigs or may alternatively bepets such as cats or dogs. Alternatively a sample of plant origin istypically obtained from a commercial crop, such as a cereal, legume,fruit or vegetable, for example wheat, barley, oats, canola, maize,soya, rice, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans,lentils, sugar cane, cocoa, cotton.

The sample may be a non-biological sample. The non-biological sample ispreferably a fluid sample. Examples of a non-biological sample includesurgical fluids, water such as drinking water, sea water or river water,and reagents for laboratory tests.

The sample is typically processed prior to being assayed, for example bycentrifugation or by passage through a membrane that filters outunwanted molecules or cells, such as red blood cells. The sample may bemeasured immediately upon being taken. The sample may also be typicallystored prior to assay, preferably below −70° C.

A transmembrane pore is a structure that permits hydrated ions driven byan applied potential to flow from one side of the membrane to the otherside of the membrane.

Any membrane may be used in accordance with the invention. Suitablemembranes are well-known in the art. The membrane is preferably anamphiphilic layer. An amphiphilic layer is a layer formed fromamphiphilic molecules, such as phospholipids, which have bothhydrophilic and lipophilic properties. The amphiphilic layer may be amonolayer or a bilayer.

The membrane is preferably a lipid bilayer. Lipid bilayers are models ofcell membranes and serve as excellent platforms for a range ofexperimental studies. For example, lipid bilayers can be used for invitro investigation of membrane proteins by single-channel recording.Alternatively, lipid bilayers can be used as biosensors to detect thepresence of a range of substances. The lipid bilayer may be any lipidbilayer. Suitable lipid bilayers include, but are not limited to, aplanar lipid bilayer, a supported bilayer or a liposome. The lipidbilayer is preferably a planar lipid bilayer. Suitable lipid bilayersare disclosed in International Application No. PCT/GB08/000563(published as WO 2008/102121), International Application No.PCT/GB08/004127 (published as WO 2009/077734) and InternationalApplication No. PCT/GB2006/001057 (published as WO 2006/100484).

Methods for forming lipid bilayers are known in the art. Suitablemethods are disclosed in the Example. Lipid bilayers are commonly formedby the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972;69: 3561-3566), in which a lipid monolayer is carried on aqueoussolution/air interface past either side of an aperture which isperpendicular to that interface.

The method of Montal & Mueller is popular because it is a cost-effectiveand relatively straightforward method of forming good quality lipidbilayers that are suitable for protein pore insertion. Other commonmethods of bilayer formation include tip-dipping, painting bilayers andpatch-clamping of liposome bilayers.

In a preferred embodiment, the lipid bilayer is formed as described inInternational Application No. PCT/GB08/004127 (published as WO2009/077734).

In another preferred embodiment, the membrane is a solid state layer. Asolid-state layer is not of biological origin. In other words, a solidstate layer is not derived from or isolated from a biologicalenvironment such as an organism or cell, or a synthetically manufacturedversion of a biologically available structure. Solid state layers can beformed from both organic and inorganic materials including, but notlimited to, microelectronic materials, insulating materials such asSi₃N₄, Al₂O₃, and SiO, organic and inorganic polymers such as polyamide,plastics such as Teflon® or elastomers such as two-componentaddition-cure silicone rubber, and glasses. The solid state layer may beformed from monatomic layers, such as graphene, or layers that are onlya few atoms thick. Suitable graphene layers are disclosed inInternational Application No. PCT/US2008/010637 (published as WO2009/035647).

The method is typically carried out using (i) an artificial bilayercomprising a pore, (ii) an isolated, naturally-occurring lipid bilayercomprising a pore, or (iii) a cell having a pore inserted therein. Themethod is preferably carried out using an artificial lipid bilayer. Thebilayer may comprise other transmembrane and/or intramembrane proteinsas well as other molecules in addition to the pore. Suitable apparatusand conditions are discussed below. The method of the invention istypically carried out in vitro.

The polynucleotide may be coupled to the membrane. This may be doneusing any known method. If the membrane is an amphiphilic layer, such asa lipid bilayer (as discussed in detail above), the polynucleotide ispreferably coupled to the membrane via a polypeptide present in themembrane or a hydrophobic anchor present in the membrane. Thehydrophobic anchor is preferably a lipid, fatty acid, sterol, carbonnanotube or amino acid.

The polynucleotide may be coupled directly to the membrane. Thepolynucleotide is preferably coupled to the membrane via a linker.Preferred linkers include, but are not limited to, polymers, such aspolynucleotides, polyethylene glycols (PEGs) and polypeptides. If apolynucleotide is coupled directly to the membrane, then some data willbe lost as the characterising run cannot continue to the end of thepolynucleotide due to the distance between the membrane and thehelicase. If a linker is used, then the polynucleotide can be processedto completion. If a linker is used, the linker may be attached to thepolynucleotide at any position. The linker is preferably attached to thepolynucleotide at the tail polymer.

The coupling may be stable or transient. For certain applications, thetransient nature of the coupling is preferred. If a stable couplingmolecule were attached directly to either the 5′ or 3′ end of apolynucleotide, then some data will be lost as the characterising runcannot continue to the end of the polynucleotide due to the distancebetween the bilayer and the helicase's active site. If the coupling istransient, then when the coupled end randomly becomes free of thebilayer, then the polynucleotide can be processed to completion.Chemical groups that form stable or transient links with the membraneare discussed in more detail below. The polynucleotide may betransiently coupled to an amphiphilic layer or lipid bilayer usingcholesterol or a fatty acyl chain. Any fatty acyl chain having a lengthof from 6 to 30 carbon atoms, such as hexadecanoic acid, may be used.

In preferred embodiments, polynucleotide is coupled to a lipid bilayer.Coupling of polynucleotides to synthetic lipid bilayers has been carriedout previously with various different tethering strategies. These aresummarised in Table 1 below.

TABLE 1 Attachment Type of group coupling Reference Thiol StableYoshina-Ishii, C. and S. G. Boxer (2003). “Arrays of mobile tetheredvesicles on supported lipid bilayers.” J Am Chem Soc 125(13): 3696-7.Biotin Stable Nikolov, V., R. Lipowsky, et al. (2007). “Behavior ofgiant vesicles with anchored DNA molecules.” Biophys J 92(12): 4356-68Cholestrol Transient Pfeiffer, I. and F. Hook (2004). “Bivalentcholesterol-based coupling of oligonucletides to lipid membraneassemblies.” J Am Chem Soc 126(33): 10224-5 Lipid Stable van Lengerich,B., R. J. Rawle, et al. “Covalent attachment of lipid vesicles to afluid-supported bilayer allows observation of DNA-mediated vesicleinteractions.” Langmuir 26(11): 8666-72

Polynucleotides may be functionalized using a modified phosphoramiditein the synthesis reaction, which is easily compatible for the additionof reactive groups, such as thiol, cholesterol, lipid and biotin groups.These different attachment chemistries give a suite of attachmentoptions for polynucleotides. Each different modification group tethersthe polynucleotide in a slightly different way and coupling is notalways permanent so giving different dwell times for the polynucleotideto the bilayer. The advantages of transient coupling are discussedabove.

Coupling of polynucleotides can also be achieved by a number of othermeans provided that a reactive group can be added to the polynucleotide.The addition of reactive groups to either end of DNA has been reportedpreviously. A thiol group can be added to the 5′ of ssDNA usingpolynucleotide kinase and ATPγS (Grant, G. P. and P. Z. Qin (2007). “Afacile method for attaching nitroxide spin labels at the 5′ terminus ofnucleic acids.” Nucleic Acids Res 35(10): e77). A more diverse selectionof chemical groups, such as biotin, thiols and fluorophores, can beadded using terminal transferase to incorporate modifiedoligonucleotides to the 3′ of ssDNA (Kumar, A., P. Tchen, et al. (1988).“Nonradioactive labeling of synthetic oligonucleotide probes withterminal deoxynucleotidyl transferase.” Anal Biochem 169(2): 376-82).

Alternatively, the reactive group could be considered to be the additionof a short piece of DNA complementary to one already coupled to thebilayer, so that attachment can be achieved via hybridisation. Ligationof short pieces of ssDNA have been reported using T4 RNA ligase I(Troutt, A. B., M. G. McHeyzer-Williams, et al. (1992).“Ligation-anchored PCR: a simple amplification technique withsingle-sided specificity.” Proc Natl Acad Sci USA 89(20): 9823-5).Alternatively either ssDNA or dsDNA could be ligated to native dsDNA andthen the two strands separated by thermal or chemical denaturation. Tonative dsDNA, it is possible to add either a piece of ssDNA to one orboth of the ends of the duplex, or dsDNA to one or both ends. Then, whenthe duplex is melted, each single strand will have either a 5′ or 3′modification if ssDNA was used for ligation or a modification at the 5′end, the 3′ end or both if dsDNA was used for ligation. If thepolynucleotide is a synthetic strand, the coupling chemistry can beincorporated during the chemical synthesis of the polynucleotide. Forinstance, the polynucleotide can be synthesized using a primer areactive group attached to it.

A common technique for the amplification of sections of genomic DNA isusing polymerase chain reaction (PCR). Here, using two syntheticoligonucleotide primers, a number of copies of the same section of DNAcan be generated, where for each copy the 5′ of each strand in theduplex will be a synthetic polynucleotide. By using an antisense primerthat has a reactive group, such as a cholesterol, thiol, biotin orlipid, each copy of the target DNA amplified will contain a reactivegroup for coupling.

The transmembrane pore is preferably a transmembrane protein pore. Atransmembrane protein pore is a polypeptide or a collection ofpolypeptides that permits hydrated ions, such as analyte, to flow fromone side of a membrane to the other side of the membrane. In the presentinvention, the transmembrane protein pore is capable of forming a porethat permits hydrated ions driven by an applied potential to flow fromone side of the membrane to the other. The transmembrane protein porepreferably permits analyte such as nucleotides to flow from one side ofthe membrane, such as a lipid bilayer, to the other. The transmembraneprotein pore allows a polynucleotide, such as DNA or RNA, to be movedthrough the pore.

The transmembrane protein pore may be a monomer or an oligomer. The poreis preferably made up of several repeating subunits, such as 6, 7 or 8subunits. The pore is more preferably a heptameric or octameric pore.

The transmembrane protein pore typically comprises a barrel or channelthrough which the ions may flow. The subunits of the pore typicallysurround a central axis and contribute strands to a transmembrane βbarrel or channel or a transmembrane α-helix bundle or channel.

The barrel or channel of the transmembrane protein pore typicallycomprises amino acids that facilitate interaction with analyte, such asnucleotides, polynucleotides or nucleic acids. These amino acids arepreferably located near a constriction of the barrel or channel. Thetransmembrane protein pore typically comprises one or more positivelycharged amino acids, such as arginine, lysine or histidine, or aromaticamino acids, such as tyrosine or tryptophan. These amino acids typicallyfacilitate the interaction between the pore and nucleotides,polynucleotides or nucleic acids.

Transmembrane protein pores for use in accordance with the invention canbe derived from β-barrel pores or α-helix bundle pores. β-barrel porescomprise a barrel or channel that is formed from β-strands. Suitableβ-barrel pores include, but are not limited to, β-toxins, such asα-hemolysin, anthrax toxin and leukocidins, and outer membraneproteins/porins of bacteria, such as Mycobacterium smegmatis porin(Msp), for example MspA, outer membrane porin F (OmpF), outer membraneporin G (OmpG), outer membrane phospholipase A and Neisseriaautotransporter lipoprotein (NalP). α-helix bundle pores comprise abarrel or channel that is formed from α-helices. Suitable α-helix bundlepores include, but are not limited to, inner membrane proteins and aouter membrane proteins, such as WZA and ClyA toxin. The transmembranepore may be derived from Msp or from α-hemolysin (α-HL).

The transmembrane protein pore is preferably derived from Msp,preferably from MspA. Such a pore will be oligomeric and typicallycomprises 7, 8, 9 or 10 monomers derived from Msp. The pore may be ahomo-oligomeric pore derived from Msp comprising identical monomers.Alternatively, the pore may be a hetero-oligomeric pore derived from Mspcomprising at least one monomer that differs from the others. Preferablythe pore is derived from MspA or a homolog or paralog thereof.

A monomer derived from Msp comprises the sequence shown in SEQ ID NO: 2or a variant thereof. SEQ ID NO: 2 is the MS-(B1)8 mutant of the MspAmonomer. It includes the following mutations: D90N, D91N, D93N, D118R,D134R and E139K. A variant of SEQ ID NO: 2 is a polypeptide that has anamino acid sequence which varies from that of SEQ ID NO: 2 and whichretains its ability to form a pore. The ability of a variant to form apore can be assayed using any method known in the art. For instance, thevariant may be inserted into a lipid bilayer along with otherappropriate subunits and its ability to oligomerise to form a pore maybe determined. Methods are known in the art for inserting subunits intomembranes, such as lipid bilayers. For example, subunits may besuspended in a purified form in a solution containing a lipid bilayersuch that it diffuses to the lipid bilayer and is inserted by binding tothe lipid bilayer and assembling into a functional state. Alternatively,subunits may be directly inserted into the membrane using the “pick andplace” method described in M. A. Holden, H. Bayley. J. Am. Chem. Soc.2005, 127, 6502-6503 and International Application No. PCT/GB2006/001057(published as WO 2006/100484).

Over the entire length of the amino acid sequence of SEQ ID NO: 2, avariant will preferably be at least 50% homologous to that sequencebased on amino acid identity. More preferably, the variant may be atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90% and more preferably at least 95%,97% or 99% homologous based on amino acid identity to the amino acidsequence of SEQ ID NO: 2 over the entire sequence. There may be at least80%, for example at least 85%, 90% or 95%, amino acid identity over astretch of 100 or more, for example 125, 150, 175 or 200 or more,contiguous amino acids (“hard homology”).

Standard methods in the art may be used to determine homology. Forexample the UWGCG Package provides the BESTFIT program which can be usedto calculate homology, for example used on its default settings(Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUPand BLAST algorithms can be used to calculate homology or line upsequences (such as identifying equivalent residues or correspondingsequences (typically on their default settings)), for example asdescribed in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. Fet al (1990) J Mol Biol 215:403-10. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (ncbi.nlm.nih.gov/).

SEQ ID NO: 2 is the MS-(B1)8 mutant of the MspA monomer. The variant maycomprise any of the mutations in the MspB, C or D monomers compared withMspA. The mature forms of MspB, C and D are shown in SEQ ID NOs: 5 to 7.In particular, the variant may comprise the following substitutionpresent in MspB: A138P. The variant may comprise one or more of thefollowing substitutions present in MspC: A96G, N₁₀₂E and A138P. Thevariant may comprise one or more of the following mutations present inMspD: Deletion of G1, L2V, ESQ, L8V, D13G, W21A, D22E, K47T, I49H, I68V,D91G, A96Q, N102D, S103T, V1041, S136K and G141A. The variant maycomprise combinations of one or more of the mutations and substitutionsfrom Msp B, C and D. The variant preferably comprises the mutation L88N.The variant of SEQ ID NO: 2 has the mutation L88N in addition to all themutations of MS-B1 and is called MS-B2. The pore used in the inventionis preferably MS-(B2)8.

Amino acid substitutions may be made to the amino acid sequence of SEQID NO: 2 in addition to those discussed above, for example up to 1, 2,3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replaceamino acids with other amino acids of similar chemical structure,similar chemical properties or similar side-chain volume. The aminoacids introduced may have similar polarity, hydrophilicity,hydrophobicity, basicity, acidity, neutrality or charge to the aminoacids they replace. Alternatively, the conservative substitution mayintroduce another amino acid that is aromatic or aliphatic in the placeof a pre-existing aromatic or aliphatic amino acid. Conservative aminoacid changes are well-known in the art and may be selected in accordancewith the properties of the 20 main amino acids as defined in Table 2below. Where amino acids have similar polarity, this can also bedetermined by reference to the hydropathy scale for amino acid sidechains in Table 3.

TABLE 2 Chemical properties of amino acids Ala aliphatic, hydrophobic,neutral Cys polar, hydrophobic, neutral Asp polar, hydrophilic, charged(−) Glu polar, hydrophilic, charged (−) Phe aromatic, hydrophobic,neutral Gly aliphatic, neutral His aromatic, polar, hydrophilic, charged(+) Ile aliphatic, hydrophobic, neutral Lys polar, hydrophilic,charged(+) Leu aliphatic, hydrophobic, neutral Met hydrophobic, neutralAsn polar, hydrophilic, neutral Pro hydrophobic, neutral Gln polar,hydrophilic, neutral Arg polar, hydrophilic, charged (+) Ser polar,hydrophilic, neutral Thr polar, hydrophilic, neutral Val aliphatic,hydrophobic, neutral Trp aromatic, hydrophobic, neutral Tyr aromatic,polar, hydrophobic

TABLE 3 Hydropathy scale Side Chain Hydropathy Ile 4.5 Val 4.2 Leu 3.8Phe 2.8 Cys 2.5 Met 1.9 Ala 1.8 Gly −0.4 Thr −0.7 Ser −0.8 Trp −0.9 Tyr−1.3 Pro −1.6 His −3.2 Glu −3.5 Gln −3.5 Asp −3.5 Asn −3.5 Lys −3.9 Arg−4.5

One or more amino acid residues of the amino acid sequence of SEQ ID NO:2 may additionally be deleted from the polypeptides described above. Upto 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may include fragments of SEQ ID NO: 2. Such fragments retainpore forming activity. Fragments may be at least 50, 100, 150 or 200amino acids in length. Such fragments may be used to produce the pores.A fragment preferably comprises the pore forming domain of SEQ ID NO: 2.Fragments must include one of residues 88, 90, 91, 105, 118 and 134 ofSEQ ID NO: 2. Typically, fragments include all of residues 88, 90, 91,105, 118 and 134 of SEQ ID NO: 2.

One or more amino acids may be alternatively or additionally added tothe polypeptides described above. An extension may be provided at theamino terminal or carboxy terminal of the amino acid sequence of SEQ IDNO: 2 or polypeptide variant or fragment thereof. The extension may bequite short, for example from 1 to 10 amino acids in length.Alternatively, the extension may be longer, for example up to 50 or 100amino acids. A carrier protein may be fused to an amino acid sequenceaccording to the invention. Other fusion proteins are discussed in moredetail below.

As discussed above, a variant is a polypeptide that has an amino acidsequence which varies from that of SEQ ID NO: 2 and which retains itsability to form a pore. A variant typically contains the regions of SEQID NO: 2 that are responsible for pore formation. The pore formingability of Msp, which contains a β-barrel, is provided by β-sheets ineach subunit. A variant of SEQ ID NO: 2 typically comprises the regionsin SEQ ID NO: 2 that form β-sheets. One or more modifications can bemade to the regions of SEQ ID NO: 2 that form β-sheets as long as theresulting variant retains its ability to form a pore. A variant of SEQID NO: 2 preferably includes one or more modifications, such assubstitutions, additions or deletions, within its α-helices and/or loopregions.

The monomers derived from Msp may be modified to assist theiridentification or purification, for example by the addition of histidineresidues (a hist tag), aspartic acid residues (an asp tag), astreptavidin tag or a flag tag, or by the addition of a signal sequenceto promote their secretion from a cell where the polypeptide does notnaturally contain such a sequence. An alternative to introducing agenetic tag is to chemically react a tag onto a native or engineeredposition on the pore. An example of this would be to react a gel-shiftreagent to a cysteine engineered on the outside of the pore. This hasbeen demonstrated as a method for separating hemolysin hetero-oligomers(Chem Biol. 1997 July; 4(7):497-505).

The monomer derived from Msp may be labelled with a revealing label. Therevealing label may be any suitable label which allows the pore to bedetected. Suitable labels include, but are not limited to, fluorescentmolecules, radioisotopes, e.g. ¹²⁵I ³⁵S, enzymes, antibodies, antigens,polynucleotides and ligands such as biotin.

The monomer derived from Msp may also be produced using D-amino acids.For instance, the monomer derived from Msp may comprise a mixture ofL-amino acids and D-amino acids. This is conventional in the art forproducing such proteins or peptides.

The monomer derived from Msp contains one or more specific modificationsto facilitate nucleotide discrimination. The monomer derived from Mspmay also contain other non-specific modifications as long as they do notinterfere with pore formation. A number of non-specific side chainmodifications are known in the art and may be made to the side chains ofthe monomer derived from Msp. Such modifications include, for example,reductive alkylation of amino acids by reaction with an aldehydefollowed by reduction with NaBH₄, amidination with methylacetimidate oracylation with acetic anhydride.

The monomer derived from Msp can be produced using standard methodsknown in the art. The monomer derived from Msp may be made syntheticallyor by recombinant means. For example, the pore may be synthesized by invitro translation and transcription (IVTT). Suitable methods forproducing pores are discussed in International Application Nos.PCT/GB09/001690 (published as WO 2010/004273), PCT/GB09/001679(published as WO 2010/004265) or PCT/GB10/000133 (published as WO2010/086603). Methods for inserting pores into membranes are discussed.

The transmembrane protein pore is also preferably derived fromα-hemolysin (α-HL). The wild type α-HL pore is formed of seven identicalmonomers or subunits (i.e. it is heptameric). The sequence of onemonomer or subunit of α-hemolysin-NN is shown in SEQ ID NO: 4. Thetransmembrane protein pore preferably comprises seven monomers eachcomprising the sequence shown in SEQ ID NO: 4 or a variant thereof.Amino acids 1, 7 to 21, 31 to 34, 45 to 51, 63 to 66, 72, 92 to 97, 104to 111, 124 to 136, 149 to 153, 160 to 164, 173 to 206, 210 to 213, 217,218, 223 to 228, 236 to 242, 262 to 265, 272 to 274, 287 to 290 and 294of SEQ ID NO: 4 form loop regions. Residues 113 and 147 of SEQ ID NO: 4form part of a constriction of the barrel or channel of α-HL.

In such embodiments, a pore comprising seven proteins or monomers eachcomprising the sequence shown in SEQ ID NO: 4 or a variant thereof arepreferably used in the method of the invention. The seven proteins maybe the same (homoheptamer) or different (heteroheptamer).

A variant of SEQ ID NO: 4 is a protein that has an amino acid sequencewhich varies from that of SEQ ID NO: 4 and which retains its poreforming ability. The ability of a variant to form a pore can be assayedusing any method known in the art. For instance, the variant may beinserted into a lipid bilayer along with other appropriate subunits andits ability to oligomerise to form a pore may be determined. Methods areknown in the art for inserting subunits into membranes, such as lipidbilayers. Suitable methods are discussed above.

The variant may include modifications that facilitate covalentattachment to or interaction with the helicase. The variant preferablycomprises one or more reactive cysteine residues that facilitateattachment to the helicase. For instance, the variant may include acysteine at one or more of positions 8, 9, 17, 18, 19, 44, 45, 50, 51,237, 239 and 287 and/or on the amino or carboxy terminus of SEQ ID NO:4. Preferred variants comprise a substitution of the residue at position8, 9, 17, 237, 239 and 287 of SEQ ID NO: 4 with cysteine (ABC, T9C,N₁₇C, K237C, S239C or E287C). The variant is preferably any one of thevariants described in International Application No. PCT/GB09/001690(published as WO 2010/004273), PCT/GB09/001679 (published as WO2010/004265) or PCT/GB10/000133 (published as WO 2010/086603).

The variant may also include modifications that facilitate anyinteraction with nucleotides.

The variant may be a naturally occurring variant which is expressednaturally by an organism, for instance by a Staphylococcus bacterium.Alternatively, the variant may be expressed in vitro or recombinantly bya bacterium such as Escherichia coli. Variants also includenon-naturally occurring variants produced by recombinant technology.Over the entire length of the amino acid sequence of SEQ ID NO: 4, avariant will preferably be at least 50% homologous to that sequencebased on amino acid identity. More preferably, the variant polypeptidemay be at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90% and more preferably atleast 95%, 97% or 99% homologous based on amino acid identity to theamino acid sequence of SEQ ID NO: 4 over the entire sequence. There maybe at least 80%, for example at least 85%, 90% or 95%, amino acididentity over a stretch of 200 or more, for example 230, 250, 270 or 280or more, contiguous amino acids (“hard homology”). Homology can bedetermined as discussed above.

Amino acid substitutions may be made to the amino acid sequence of SEQID NO: 4 in addition to those discussed above, for example up to 1, 2,3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions may bemade as discussed above.

One or more amino acid residues of the amino acid sequence of SEQ ID NO:4 may additionally be deleted from the polypeptides described above. Upto 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may be fragments of SEQ ID NO: 4. Such fragments retainpore-forming activity. Fragments may be at least 50, 100, 200 or 250amino acids in length. A fragment preferably comprises the pore-formingdomain of SEQ ID NO: 4. Fragments typically include residues 119, 121,135, 113 and 139 of SEQ ID NO: 4.

One or more amino acids may be alternatively or additionally added tothe polypeptides described above. An extension may be provided at theamino terminus or carboxy terminus of the amino acid sequence of SEQ IDNO: 4 or a variant or fragment thereof. The extension may be quiteshort, for example from 1 to 10 amino acids in length. Alternatively,the extension may be longer, for example up to 50 or 100 amino acids. Acarrier protein may be fused to a pore or variant.

As discussed above, a variant of SEQ ID NO: 4 is a subunit that has anamino acid sequence which varies from that of SEQ ID NO: 4 and whichretains its ability to form a pore. A variant typically contains theregions of SEQ ID NO: 4 that are responsible for pore formation. Thepore forming ability of α-HL, which contains a β-barrel, is provided byβ-strands in each subunit. A variant of SEQ ID NO: 4 typically comprisesthe regions in SEQ ID NO: 4 that form β-strands. The amino acids of SEQID NO: 4 that form β-strands are discussed above. One or moremodifications can be made to the regions of SEQ ID NO: 4 that formβ-strands as long as the resulting variant retains its ability to form apore. Specific modifications that can be made to the β-strand regions ofSEQ ID NO: 4 are discussed above.

A variant of SEQ ID NO: 4 preferably includes one or more modifications,such as substitutions, additions or deletions, within its α-helicesand/or loop regions. Amino acids that form α-helices and loops arediscussed above.

The variant may be modified to assist its identification or purificationas discussed above.

Pores derived from α-HL can be made as discussed above with reference topores derived from Msp.

In some embodiments, the transmembrane protein pore is chemicallymodified. The pore can be chemically modified in any way and at anysite. The transmembrane protein pore is preferably chemically modifiedby attachment of a molecule to one or more cysteines (cysteine linkage),attachment of a molecule to one or more lysines, attachment of amolecule to one or more non-natural amino acids, enzyme modification ofan epitope or modification of a terminus. Suitable methods for carryingout such modifications are well-known in the art. The transmembraneprotein pore may be chemically modified by the attachment of anymolecule. For instance, the pore may be chemically modified byattachment of a dye or a fluorophore.

Any number of the monomers in the pore may be chemically modified. Oneor more, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the monomers ispreferably chemically modified as discussed above.

The reactivity of cysteine residues may be enhanced by modification ofthe adjacent residues. For instance, the basic groups of flankingarginine, histidine or lysine residues will change the pKa of thecysteines thiol group to that of the more reactive 5⁻ group. Thereactivity of cysteine residues may be protected by thiol protectivegroups such as dTNB. These may be reacted with one or more cysteineresidues of the pore before a linker is attached.

The molecule (with which the pore is chemically modified) may beattached directly to the pore or attached via a linker as disclosed inInternational Application Nos. PCT/GB09/001690 (published as WO2010/004273), PCT/GB09/001679 (published as WO 2010/004265) orPCT/GB10/000133 (published as WO 2010/086603).

Any Hel308 helicase may be used in accordance with the invention. Hel308helicases are also known as ski2-like helicases and the two terms can beused interchangeably.

The Hel308 helicase typically comprises the amino acid motifQ-X1-X2-G-R-A-G-R (hereinafter called the Hel308 motif; SEQ ID NO: 8).The Hel308 motif is typically part of the helicase motif VI (Tuteja andTuteja, Eur. J. Biochem. 271, 1849-1863 (2004)). X1 may be C, M or L. X1is preferably C. X2 may be any amino acid residue. X2 is typically ahydrophobic or neutral residue. X2 may be A, F, M, C, V, L, I, S, T, Por R. X2 is preferably A, F, M, C, V, L, I, S, T or P. X2 is morepreferably A, M or L. X2 is most preferably A or M.

The Hel308 helicase preferably comprises the motif Q-X1-X2 GR A GRP(hereinafter called the extended Hel308 motif; SEQ ID NO: 9) wherein X1and X2 are as described above.

The most preferred Hel308 motifs and extended Hel308 motifs are shown inTable 5 below. The Hel308 helicase may comprise any of these preferredmotifs.

The Hel308 helicase is preferably one of the helicases shown in Table 4below or a variant thereof.

TABLE 4 Preferred Hel308 helicases Accession Description NP_578406.1ski2-like helicase [Pyrococcus furiosus DSM3638] >sp|O73946.1|HELS_PYRFU RecName: Full = Putative ski2-typehelicase >pdb|2ZJ2|A Chain A, Archaeal Dna Helicase Hjm Apo State InForm 1 >pdb|2ZJ5|A Chain A, Archaeal Dna Helicase Hjm Complexed With AdpIn Form 1 >pdb|2ZJ8|A Chain A, Archaeal Dna Helicase Hjm Apo State InForm 2 >pdb|2ZJA|A Chain A, Archaeal Dna Helicase Hjm Complexed WithAmppcp In Form 2 >dbj|BAA32016.1| helicase [Pyrococcusfuriosus] >gb|AAL80801.1| helicase [Pyrococcus furiosus DSM 3638]NP_126564.1 ski2-like helicase [Pyrococcus abyssiGE5] >sp|Q9V0A9.1|HELS_PYRAB RecName: Full = Putative ski2-typehelicase >emb|CAB49795.1| DNA helicase [Pyrococcus abyssi GE5]NP_143168.1 ski2-like helicase [Pyrococcus horikoshiiOT3] >sp|O59025.1|HELS_PYRHO RecName: Full = Putative ski2-typehelicase >dbj|BAA30383.1| 715aa long hypothetical protein [Pyrococcushorikoshii OT3] YP_004424773.1 ski2-like helicase [Pyrococcus sp.NA2] >gb|AEC52769.1| ski2-like helicase [Pyrococcus sp. NA2]YP_004623750.1 ski2-like helicase [Pyrococcus yayanosiiCH1] >gb|AEH24478.1| ski2-like helicase [Pyrococcus yayanosii CH1]YP_002307730.1 ski2-like helicase [Thermococcus onnurineusNA1] >gb|ACJ16833.1| DNA helicase [Thermococcus onnurineus NA1]YP_004763427.1 ski2-like helicase [Thermococcus sp.4557] >gb|AEK73750.1| ski2-like helicase [Thermococcus sp. 4557]YP_002959236.1 ski2-like helicase [Thermococcus gammatoleransEJ3] >gb|ACS33372.1| ski2- type helicase, putative [Thermococcusgammatolerans EJ3] YP_004071709.1 ski2-type helicase [Thermococcusbarophilus MP] >gb|ADT84486.1| putative ski2-type helicase [Thermococcusbarophilus MP] YP_002994328.1 Putative ski2-type helicase [Thermococcussibiricus MM 739] >gb|ACS89979.1| Putative ski2-type helicase[Thermococcus sibiricus MM 739] ZP_04875329.1 Type III restrictionenzyme, res subunit family [Aciduliprofundum booneiT469] >gb|EDY35111.1| Type III restriction enzyme, res subunit family[Aciduliprofundum boonei T469] YP_003436565.1 DEAD/DEAH box helicase[Ferroglobus placidus DSM 10642] >gb|ADC66290.1| DEAD/DEAH box helicasedomain protein [Ferroglobus placidus DSM 10642] YP_004485304.1 ski2-typehelicase [Methanotorris igneus Kol 5] >gb|AEF97239.1| ski2-type helicase[Methanotorris igneus Kol 5] YP_004616424.1 DEAD/DEAH box helicasedomain-containing protein [Methanosalsum zhilinae DSM4017] >gb|AEH61205.1| DEAD/DEAH box helicase domain protein[Methanosalsum zhilinae DSM 4017] ZP_04873370.1 Type III restrictionenzyme, res subunit family [Aciduliprofundum booneiT469] >ref|YP_003482774.1| DEAD/DEAH box helicase domain protein[Aciduliprofundum boonei T469] >gb|EDY36687.1| Type III restrictionenzyme, res subunit family [Aciduliprofundum booneiT469] >gb|ADD08212.1| DEAD/DEAH box helicase domain protein[Aciduliprofundum boonei T469] YP_004342552.1 ski2-type helicase[Archaeoglobus veneficus SNP6] >gb|AEA47837.1| ski2-type helicase[Archaeoglobus veneficus SNP6] NP_071282.1 SKI2-family helicase[Archaeoglobus fulgidus DSM 4304] 2P6R_A Chain A, Crystal Structure OfSuperfamily 2 Helicase Hel308 In Complex With Unwound Dna >pdb|2P6U|AChain A, Apo Structure Of The Hel308 Superfamily 2 Helicase YP_685308.1ski2-like helicase [uncultured methanogenic archaeonRC-I] >sp|Q0W6L1.1|HELS_UNCMA RecName: Full = Putative ski2-typehelicase >emb|CAJ35982.1| putative ski2-type helicase [unculturedmethanogenic archaeon RC-I] YP_001048404.1 ski2-like helicase[Methanoculleus marisnigri JR1] >gb|ABN58422.1| DEAD/DEAH box helicasedomain protein [Methanoculleus marisnigri JR1] YP_919908.1 DEAD/DEAH boxhelicase domain-containing protein [Thermofilum pendens Hrk5] >gb|ABL77905.1| DEAD/DEAH box helicase domain protein [Thermofilumpendens Hrk 5] YP_843229.1 ski2-like helicase [Methanosaeta thermophilaPT] >gb|ABK14589.1| DEAD/DEAH box helicase domain protein [Methanosaetathermophila PT] ZP_08045937.1 ski2-like helicase [Haladaptatuspaucihalophilus DX253] >gb|EFW90585.1| ski2- like helicase [Haladaptatuspaucihalophilus DX253] NP_280985.1 ski2-like helicase [Halobacterium sp.NRC-1] >ref|YP_001690117.1| ski2-like helicase [Halobacterium salinarumR1] >sp|Q9HMV6.1|HELS_HALSA RecName: Full = Putative ski2-typehelicase >sp|B0R7Q2.1|HELS_HALS3 RecName: Full = Putative ski2-typehelicase >gb|AAG20465.1| DNA repair protein [Halobacterium sp.NRC-1] >emb|CAP14771.1| putative DNA helicase [Halobacterium salinarumR1] YP_003357840.1 Holliday junction migration helicase [Methanocellapaludicola SANAE] >dbj|BAI62857.1| Holliday junction migration helicase[Methanocella paludicola SANAE] YP_003457479.1 DEAD/DEAH box helicasedomain protein [Methanocaldococcus sp. FS406-22] >gb|ADC68743.1|DEAD/DEAH box helicase domain protein [Methanocaldococcus sp. FS406-22]YP_003127632.1 DEAD/DEAH box helicase domain protein [Methanocaldococcusfervens AG86] >gb|ACV24132.1| DEAD/DEAH box helicase domain protein[Methanocaldococcus fervens AG86] YP_003735335.1 ski2-like helicase[Halalkalicoccus jeotgali B3] >gb|ADJ13543.1| ski2-like helicase[Halalkalicoccus jeotgali B3] YP_503885.1 ski2-like helicase[Methanospirillum hungatei JF-1] >gb|ABD42166.1| DEAD/DEAH boxhelicase-like protein [Methanospirillum hungatei JF-1] BAJ48115.1helicase [Candidates Caldiarchaeum subterraneum] >dbj|BAJ48144.1|helicase [Candidates Caldiarchaeum subterraneum] >dbj|BAJ50919.1|helicase [Candidates Caldiarchaeum subterraneum] YP_001405615.1ski2-like helicase [Candidates Methanoregula boonei6A8] >sp|A7IB61.1|HELS_METB6 RecName: Full = Putative ski2-typehelicase >gb|ABS56972.1| DEAD/DEAH box helicase domain protein[Methanoregula boonei 6A8] YP_306959.1 ski2-like helicase[Methanosarcina barkeri str. Fusaro] >sp|Q465R3.1|HELS_METBF RecName:Full = Putative ski2-type helicase >gb|AAZ72379.1| helicase[Methanosarcina barkeri str. Fusaro] YP_001031179.1 ski2-like helicase[Methanocorpusculum labreanum Z] >gb|ABN07912.1| DEAD/DEAH box helicasedomain protein [Methanocorpusculum labreanum Z] YP_003541733.1 DEAD/DEAHbox helicase [Methanohalophilus mahii DSM 5219] >gb|ADE36088.1|DEAD/DEAH box helicase domain protein [Methanohalophilus mahii DSM 5219]YP_004384692.1 putative Ski2-type helicase [Methanosaeta conciliiGP6] >gb|AEB68874.1| putative Ski2-type helicase [Methanosaeta conciliiGP6] YP_003725904.1 DEAD/DEAH box helicase domain-containing protein[Methanohalobium evestigatum Z-7303] >gb|ADI73108.1| DEAD/DEAH boxhelicase domain protein [Methanohalobium evestigatum Z-7303]YP_003405271.1 DEAD/DEAH box helicase [Haloterrigena turkmenica DSM5511] >gb|ADB62598.1| DEAD/DEAH box helicase domain protein[Haloterrigena turkmenica DSM 5511] YP_004244914.1 DEAD/DEAH boxhelicase [Vulcanisaeta moutnovskia 768-28] >gb|ADY01412.1| DEAD/DEAH boxhelicase domain protein [Vulcanisaeta moutnovskia 768-28] YP_001540156.1DEAD/DEAH box helicase domain-containing protein [Caldivirgamaquilingensis IC-167] >sp|A8MB76.1|HELS_CALMQ RecName: Full = Putativeski2-type helicase >gb|ABW01166.1| DEAD/DEAH box helicase domain protein[Caldivirga maquilingensis IC-167] NP_618094.1 ski2-like helicase[Methanosarcina acetivorans C2A] >sp|Q8TL39.1|HELS_METAC RecName: Full =Putative ski2-type helicase >gb|AAM06574.1| helicase [Methanosarcinaacetivorans C2A] YP_003900980.1 DEAD/DEAH box helicase domain-containingprotein [Vulcanisaeta distributa DSM 14429] >gb|ADN49929.1| DEAD/DEAHbox helicase domain protein [Vulcanisaeta distributa DSM 14429]YP_003896003.1 DEAD/DEAH box helicase domain-containing protein[Methanoplanus petrolearius DSM 11571] >gb|ADN37565.1| DEAD/DEAH boxhelicase domain protein [Methanoplanus petrolearius DSM 11571]YP_003615773.1 DEAD/DEAH box helicase domain protein [Methanocaldococcusinfernus ME] >gb|ADG12809.1| DEAD/DEAH box helicase domain protein[Methanocaldococcus infernus ME] YP_183745.1 RNA helicase Ski2-likeprotein [Thermococcus kodakarensis KOD1] >sp|Q5JGV6.1|HELS_PYRKORecName: Full = Putative ski2-type helicase; Contains: RecName: Full =Endonuclease PI-PkoHel; AltName: Full = Pko Hel intein >dbj|BAD85521.1|RNA helicase Ski2 homolog [Thermococcus kodakarensis KOD1]YP_001322557.1 DEAD/DEAH box helicase domain-containing protein[Methanococcus vannielii SB] >sp|A6UN73.1|HELS_METVS RecName: Full =Putative ski2-type helicase >gb|ABR53945.1| DEAD/DEAH box helicasedomain protein [Methanococcus vannielii SB] YP_002467772.1 ski2-likehelicase [Methanosphaerula palustris E1-9c] >gb|ACL18049.1| DEAD/DEAHbox helicase domain protein [Methanosphaerula palustris E1-9c]YP_003480097.1 DEAD/DEAH box helicase [Natrialba magadii ATCC43099] >gb|ADD05535.1| DEAD/DEAH box helicase domain protein [Natrialbamagadii ATCC 43099] YP_004577043.1 ski2-type helicase[Methanothermococcus okinawensis IH1] >gb|AEH07265.1| ski2-type helicase[Methanothermococcus okinawensis IH1] YP_004742641.1 superfamily IIhelicase [Methanococcus maripaludis XI] >gb|AEK19898.1| superfamily IIhelicase [Methanococcus maripaludis X1] NP_632449.1 ski2-like helicase[Methanosarcina mazei Go1] >sp|Q8PZR7.1|HELS_METMA RecName: Full =Putative ski2-type helicase >gb|AAM30121.1| helicase [Methanosarcinamazei Go1] YP_001097223.1 DEAD/DEAH box helicase domain-containingprotein [Methanococcus maripaludis C5] >gb|ABO35008.1| DEAD/DEAH boxhelicase domain protein [Methanococcus maripaludis C5] YP_004742247.1DEAD/DEAH box helicase domain-containing protein [Methanococcusmaripaludis XI] >gb|AEK19504.1| DEAD/DEAH box helicase domain-containingprotein [Methanococcus maripaludis X1] YP_004794766.1 ski2-like helicase[Haloarcula hispanica ATCC 33960] >gb|AEM55778.1| ski2- like helicase[Haloarcula hispanica ATCC 33960] NP_988010.1 superfamily II helicase[Methanococcus maripaludis S2] >emb|CAF30446.1| superfamily II helicase[Methanococcus maripaludis S2] YP_565780.1 ski2-like helicase[Methanococcoides burtonii DSM 6242] >sp|Q12WZ6.1|HELS_METBU RecName:Full = Putative ski2-type helicase >gb|ABE52030.1| DEAD/DEAH boxhelicase-like protein [Methanococcoides burtonii DSM 6242]YP_001549808.1 DEAD/DEAH box helicase domain-containing protein[Methanococcus maripaludis C6] >gb|ABX02576.1| DEAD/DEAH box helicasedomain protein [Methanococcus maripaludis C6] YP_001548609.1 DEAD/DEAHbox helicase domain-containing protein [Methanococcus maripaludisC6] >gb|ABX01377.1| DEAD/DEAH box helicase domain protein [Methanococcusmaripaludis C6] YP_001329359.1 DEAD/DEAH box helicase domain-containingprotein [Methanococcus maripaludis C7] >gb|ABR65208.1| DEAD/DEAH boxhelicase domain protein [Methanococcus maripaludis C7] YP_004595982.1ski2-type helicase [Halopiger xanaduensis SH-6] >gb|AEH36103.1|ski2-type helicase [Halopiger xanaduensis SH-6] YP_656795.1 ski2-likehelicase [Haloquadratum walsbyi DSM 16790] >emb|CAJ51138.1|ATP-dependent DNA helicase [Haloquadratum walsbyi DSM 16790] CCC38992.1ATP-dependent DNA helicase Hel308 [Haloquadratum walsbyi C23]YP_004035272.1 superfamily ii helicase [Halogeometricum borinquense DSM11551] >gb|ADQ65833.1| superfamily II helicase [Halogeometricumborinquense DSM 11551] YP_137330.1 ski2-like helicase [Haloarculamarismortui ATCC 43049] >sp|Q5UYM9.1|HELS_HALMA RecName: Full = Putativeski2-type helicase >gb|AAV47624.1| putative ski2-type helicase[Haloarcula marismortui ATCC 43049] YP_001581577.1 DEAD/DEAH boxhelicase domain-containing protein [Nitrosopumilus maritimusSCM1] >gb|ABX12139.1| DEAD/DEAH box helicase domain protein[Nitrosopumilus maritimus SCM1] EET90255.1 DEAD/DEAH box helicase domainprotein [Candidates Micrarchaeum acidiphilum ARMAN-2] NP_376477.1helicase [Sulfolobus tokodaii str. 7] >sp|Q974S1.1|HELS_SULTO RecName:Full = Putative ski2-type helicase >dbj|BAK54341.1| Holliday junctionmigration helicase [Sulfolobus tokodaii str. 7] YP_001097792.1 DEAD/DEAHbox helicase domain-containing protein [Methanococcus maripaludisC5] >gb|ABO35578.1| DEAD/DEAH box helicase domain protein [Methanococcusmaripaludis C5] ZP_08667240.1 DEAD/DEAH box helicase domain protein[Nitrosopumilus sp. MY1] >gb|EGP92972.1| DEAD/DEAH box helicase domainprotein [Nitrosopumilus sp. MY1] YP_254972.1 DNA helicase [Sulfolobusacidocaldarius DSM 639] >sp|Q4JC00.1|HELS_SULAC RecName: Full = Putativeski2-type helicase >gb|AAY79679.1| DNA helicase [Sulfolobusacidocaldarius DSM 639] EFD92533.1 DEAD/DEAH box helicase domain protein[Candidates Parvarchaeum acidophilus ARMAN-5] YP_003176527.1 ski2-likehelicase [Halomicrobium mukohataei DSM 12286] >gb|ACV46820.1| DEAD/DEAHbox helicase domain protein [Halomicrobium mukohataei DSM 12286]EGD71904.1 DEAD/DEAH box helicase domain protein [CandidatesParvarchaeum acidophilus ARMAN-5_′5-way FS′] YP_001040230.1 DEAD/DEAHbox helicase domain-containing protein [Staphylothermus marinusF1] >gb|ABN69322.1| DEAD/DEAH box helicase domain protein[Staphylothermus marinus F1] ABZ07376.1 putative DEAD/DEAH box helicase[uncultured marine crenarchaeote HF4000_ANIW133M9] YP_001097458.1DEAD/DEAH box helicase domain-containing protein [Methanococcusmaripaludis C5] >gb|ABO35243.1| DEAD/DEAH box helicase domain protein[Methanococcus maripaludis C5] ABZ08606.1 putative DEAD/DEAH boxhelicase [uncultured marine crenarchaeote HF4000_APKG3H9] YP_325906.1ski2-like helicase [Natronomonas pharaonis DSM2160] >sp|Q3IU46.1|HELS_NATPD RecName: Full = Putative ski2-typehelicase >emb|CAI48337.1| ATP-dependent DNA helicase 1 [Natronomonaspharaonis DSM 2160] YP_930665.1 DEAD/DEAH box helicase domain-containingprotein [Pyrobaculum islandicum DSM 4184] >gb|ABL88322.1| DEAD/DEAH boxhelicase domain protein [Pyrobaculum islandicum DSM 4184] YP_001435870.1DEAD/DEAH box helicase [Ignicoccus hospitalis KIN4/I] >gb|ABU82463.1|DEAD/DEAH box helicase domain protein [Ignicoccus hospitalis KIN4/I]YP_003668634.1 DEAD/DEAH box helicase domain-containing protein[Staphylothermus hellenicus DSM 12710] >gb|ADI31735.1| DEAD/DEAH boxhelicase domain protein [Staphylothermus hellenicus DSM 12710]ZP_08558598.1 ski2-like helicase [Halorhabdus tiamateaSARL4B] >gb|EGM36528.1| ski2-like helicase [Halorhabdus tiamatea SARL4B]YP_002428409.1 DEAD/DEAH box helicase domain-containing protein[Desulfurococcus kamchatkensis 1221n] >gb|ACL11042.1| DEAD/DEAH boxhelicase domain protein [Desulfurococcus kamchatkensis 1221n]YP_004336918.1 ATP-dependent, DNA binding helicase [Thermoproteusuzoniensis 768-20] >gb|AEA11606.1| ATP-dependent, DNA binding helicase[Thermoproteus uzoniensis 768-20] ZP_08257442.1 DEAD/DEAH box helicasedomain-containing protein [Candidatus Nitrosoarchaeum limniaSFB1] >gb|EGG41989.1| DEAD/DEAH box helicase domain-containing protein[Candidatus Nitrosoarchaeum limnia SFB1] YP_004459284.1 DEAD/DEAH boxhelicase domain-containing protein [Acidianus hospitalisW1] >gb|AEE94986.1| DEAD/DEAH box helicase domain protein [Acidianushospitalis W1] NP_558924.1 ATP-dependent, DNA binding helicase[Pyrobaculum aerophilum str. IM2] >gb|AAL63106.1| ATP-dependent, DNAbinding helicase [Pyrobaculum aerophilum str. IM2] YP_004409449.1DEAD/DEAH box helicase domain-containing protein [Metallosphaera cuprinaAr-4] >gb|AEB94965.1| DEAD/DEAH box helicase domain-containing protein[Metallosphaera cuprina Ar-4] YP_003649556.1 DEAD/DEAH box helicasedomain-containing protein [Thermosphaera aggregans DSM11486] >gb|ADG90604.1| DEAD/DEAH box helicase domain protein[Thermosphaera aggregans DSM 11486] ZP_06387115.1 DEAD/DEAH box helicasedomain protein [Sulfolobus solfataricus 98/2] >gb|ACX90562.1| DEAD/DEAHbox helicase domain protein [Sulfolobus solfataricus 98/2] 2VA8_A ChainA, Dna Repair Helicase Hel308 >pdb|2VA8|B Chain B, Dna Repair HelicaseHel308 >emb|CAO85626.1| DNA helicase [Sulfolobus solfataricus]YP_004809267.1 ski2-type helicase [Halophilic archaeonDL31] >gb|AEN06894.1| ski2-type helicase [Halophilic archaeon DL31]ADX84345.1 DEAD/DEAH box helicase domain protein [Sulfolobus islandicusREY15A] >gb|ADX81629.1| DEAD/DEAH box helicase domain protein[Sulfolobus islandicus HVE10/4] YP_002828439.1 DEAD/DEAH box helicase[Sulfolobus islandicus M.14.25] >ref|YP_002842325.1| DEAD/DEAH boxhelicase domain protein [Sulfolobus islandicus M.16.27] >gb|ACP37141.1|DEAD/DEAH box helicase domain protein [Sulfolobus islandicusM.14.25] >gb|ACP54280.1| DEAD/DEAH box helicase domain protein[Sulfolobus islandicus M.16.27] YP_002913571.1 DEAD/DEAH box helicasedomain protein [Sulfolobus islandicus M.16.4] >gb|ACR40903.1| DEAD/DEAHbox helicase domain protein [Sulfolobus islandicus M.16.4] Q97VY9.1RecName: Full = Putative ski2-type helicase YP_002841682.1 DEAD/DEAH boxhelicase domain protein [Sulfolobus islandicusY.N.15.51] >gb|ACP49760.1| DEAD/DEAH box helicase domain protein[Sulfolobus islandicus Y.N.15.51] YP_002831080.1 DEAD/DEAH box helicasedomain protein [Sulfolobus islandicus L.S.2.15] >ref|YP_003418425.1|DEAD/DEAH box helicase domain protein [Sulfolobus islandicusL.D.8.5] >gb|ACP34435.1| DEAD/DEAH box helicase domain protein[Sulfolobus islandicus L.S.2.15] >gb|ADB86055.1| DEAD/DEAH box helicasedomain protein [Sulfolobus islandicus L.D.8.5] YP_001054984.1 DEAD/DEAHbox helicase domain-containing protein [Pyrobaculum calidifontis JCM11548] >sp|A3MSA1.1|HELS_PYRCJ RecName: Full = Putative ski2-typehelicase >gb|ABO07518.1| DEAD/DEAH box helicase domain protein[Pyrobaculum calidifontis JCM 11548] NP_343811.1 DNA helicase relatedprotein [Sulfolobus solfataricus P2] >ref|YP_002836469.1| DEAD/DEAH boxhelicase [Sulfolobus islandicus Y.G.57.14] >gb|AAK42601.1| DNA helicaserelated protein [Sulfolobus solfataricus P2] >gb|ACP44547.1| DEAD/DEAHbox helicase domain protein [Sulfolobus islandicus Y.G.57.14]YP_001152379.1 DEAD/DEAH box helicase domain-containing protein[Pyrobaculum arsenaticum DSM 13514] >gb|ABP49727.1| DEAD/DEAH boxhelicase domain protein [Pyrobaculum arsenaticum DSM 13514]YP_001191456.1 DEAD/DEAH box helicase domain-containing protein[Metallosphaera sedula DSM 5348] >gb|ABP95532.1| DEAD/DEAH box helicasedomain protein [Metallosphaera sedula DSM 5348] NP_147034.2 hollidayjunction migration helicase [Aeropyrum pernixK1] >sp|Q9YFQ8.2|HELS_AERPE RecName: Full = Putative ski2-typehelicase >dbj|BAA79103.2| holliday junction migration helicase[Aeropyrum pernix K1] YP_024158.1 ski2-like helicase [Picrophilustorridus DSM 9790] >gb|AAT43965.1| helicase involved in UV-protection[Picrophilus torridus DSM 9790] YP_003816358.1 Putative ski2-typehelicase [Acidilobus saccharovorans 345-15] >gb|ADL19327.1| Putativeski2-type helicase [Acidilobus saccharovorans 345-15] YP_003860265.1DEAD/DEAH box helicase domain protein [Ignisphaera aggregans DSM17230] >gb|ADM28385.1| DEAD/DEAH box helicase domain protein[Ignisphaera aggregans DSM 17230] NP_394295.1 ski2-like helicase[Thermoplasma acidophilum DSM 1728] >sp|Q9HJX7.1|HELS_THEAC RecName:Full = Putative ski2-type helicase >emb|CAC11964.1| DNA helicase relatedprotein [Thermoplasma acidophilum] YP_876638.1 superfamily II helicase[Cenarchaeum symbiosum A] >gb|ABK78334.1| superfamily II helicase[Cenarchaeum symbiosum A] ZP_05571398.1 ski2-like helicase [Ferroplasmaacidarmanus fer1] YP_004176252.1 DEAD/DEAH box helicasedomain-containing protein [Desulfurococcus mucosus DSM2162] >gb|ADV64770.1| DEAD/DEAH box helicase domain protein[Desulfurococcus mucosus DSM 2162] YP_001737782.1 DEAD/DEAH box helicasedomain-containing protein [Candidatus Korarchaeum cryptofilumOPF8] >gb|ACB08099.1| DEAD/DEAH box helicase domain protein [CandidatusKorarchaeum cryptofilum OPF8] EGQ40435.1 superfamily II helicase[Candidatus Nanosalinarum sp. J07AB56] YP_002567343.1 ski2-like helicase[Halorubrum lacusprofundi ATCC 49239] >gb|ACM58273.1| DEAD/DEAH boxhelicase domain protein [Halorubrum lacusprofundi ATCC 49239]YP_001793507.1 DEAD/DEAH box helicase domain-containing protein[Thermoproteus neutrophilus V24Sta] >gb|ACB39061.1| DEAD/DEAH boxhelicase domain protein [Thermoproteus neutrophilus V24Sta]YP_003534088.1 ATP-dependent DNA helicase Hel308a [Haloferax volcaniiDS2] >gb|ADE04048.1| ATP-dependent DNA helicase Hel308a [Haloferaxvolcanii DS2] YP_004037165.1 superfamily ii helicase [Halogeometricumborinquense DSM 11551] >gb|ADQ67720.1| superfamily II helicase[Halogeometricum borinquense DSM 11551] NP_111333.1 ski2-like helicase[Thermoplasma volcanium GSS1] >sp|Q97AI2.1|HELS_THEVO RecName: Full =Putative ski2-type helicase >dbj|BAB59970.1| DNA helicase [Thermoplasmavolcanium GSS1] YP_002565871.1 DEAD/DEAH box helicase [Halorubrumlacusprofundi ATCC 49239] >gb|ACM56801.1| DEAD/DEAH box helicase domainprotein [Halorubrum lacusprofundi ATCC 49239] CCC39675.1 ATP-dependentDNA helicase Hel308 [Haloquadratum walsbyi C23] YP_657401.1ATP-dependent DNA helicase [Haloquadratum walsbyi DSM16790] >emb|CAJ51759.1| ATP-dependent DNA helicase [Haloquadratumwalsbyi DSM 16790] YP_003535028.1 ATP-dependent DNA helicase Hel308b[Haloferax volcanii DS2] >gb|ADE02398.1| ATP-dependent DNA helicaseHel308b [Haloferax volcanii DS2] YP_003706863.1 DEAD/DEAH box helicasedomain-containing protein [Methanococcus voltae A3] >gb|ADI35890.1|DEAD/DEAH box helicase domain protein [Methanococcus voltae A3]ABD17736.1 helicase [Methanococcus voltae PS] NP_613398.1 superfamily IIhelicase [Methanopyrus kandleri AV19] >gb|AAM01328.1| PredictedSuperfamily II helicase [Methanopyrus kandleri AV19] CBH38575.1 putativeski2-type helicase [uncultured archaeon] EEZ93258.1 DEAD/DEAH boxhelicase domain protein [Candidates Parvarchaeum acidiphilum ARMAN-4]EGQ40350.1 superfamily II helicase [Candidates Nanosalinarum sp.J07AB56] YP_004004246.1 dead/deah box helicase domain protein[Methanothermus fervidus DSM 2088] >gb|ADP77484.1| DEAD/DEAH boxhelicase domain protein [Methanothermus fervidus DSM 2088]YP_003850109.1 helicase [Methanothermobacter marburgensis str.Marburg] >gb|ADL58796.1| predicted helicase [Methanothermobactermarburgensis str. Marburg] YP_003424423.1 DEAD/DEAH box helicasedomain-containing protein [Methanobrevibacter ruminantiumM1] >gb|ADC47531.1| DEAD/DEAH box helicase domain- containing protein[Methanobrevibacter ruminantium M1] YP_004291107.1 DEAD/DEAH boxhelicase domain-containing protein [Methanobacterium sp.AL-21] >gb|ADZ10135.1| DEAD/DEAH box helicase domain protein[Methanobacterium sp. AL-21] YP_447162.1 helicase [Methanosphaerastadtmanae DSM 3091] >gb|ABC56519.1| predicted helicase [Methanosphaerastadtmanae DSM 3091] YP_004519549.1 DEAD/DEAH box helicasedomain-containing protein [Methanobacterium sp. SWAN-1] >gb|AEG17748.1|DEAD/DEAH box helicase domain protein [Methanobacterium sp. SWAN-1]NP_275949.1 DNA helicase related protein [Methanothermobacterthermautotrophicus str. Delta H] >sp|O26901.1|HELS_METTH RecName: Full =Putative ski2-type helicase >gb|AAB85310.1| DNA helicase related protein[Methanothermobacter thermautotrophicus str. Delta H] ZP_05975717.2putative Ski2-type helicase [Methanobrevibacter smithii DSM2374] >gb|EFC93382.1| putative Ski2-type helicase [Methanobrevibactersmithii DSM 2374] ZP_03607647.1 hypothetical protein METSMIALI_00751[Methanobrevibacter smithii DSM 2375] >gb|EEE41862.1| hypotheticalprotein METSMIALI_00751 [Methanobrevibacter smithii DSM 2375]YP_001273412.1 ATP-dependent helicase [Methanobrevibacter smithii ATCC35061] >gb|ABQ87044.1| ATP-dependent helicase [Methanobrevibactersmithii ATCC 35061] YP_003247505.1 DEAD/DEAH box helicase domain protein[Methanocaldococcus vulcanius M7] >gb|ACX73023.1| DEAD/DEAH box helicasedomain protein [Methanocaldococcus vulcanius M7] NP_248116.1 SKI2 familyhelicase [Methanocaldococcus jannaschii DSM2661] >sp|Q58524.1|HELS_METJA RecName: Full = Putative ski2-typehelicase; Contains: RecName: Full = Endonuclease PI-MjaHel; AltName:Full = Mja Hel intein; AltName: Full = Mja Pep3 intein >gb|AAB99126.1|putative SKI2-family helicase [Methanocaldococcus jannaschii DSM 2661]YP_001324295.1 DEAD/DEAH box helicase domain-containing protein[Methanococcus aeolicus Nankai-3] >gb|ABR55683.1| DEAD/DEAH box helicasedomain protein [Methanococcus aeolicus Nankai-3] YP_003536960.1 Pre-mRNAsplicing helicase [Haloferax volcanii DS2] >gb|ADE02332.1| Pre- mRNAsplicing helicase [Haloferax volcanii DS2] YP_003131029.1 DEAD/DEAH boxhelicase domain protein [Halorhabdus utahensis DSM12940] >gb|ACV12296.1| DEAD/DEAH box helicase domain protein[Halorhabdus utahensis DSM 12940] YP_002567151.1 DEAD/DEAH box helicase[Halorubrum lacusprofundi ATCC 49239] >gb|ACM58081.1| DEAD/DEAH boxhelicase domain protein [Halorubrum lacusprofundi ATCC 49239]YP_004035351.1 superfamily ii helicase [Halogeometricum borinquense DSM11551] >gb|ADQ65912.1| superfamily II helicase [Halogeometricumborinquense DSM 11551] YP_004808851.1 DEAD/DEAH box helicasedomain-containing protein [Halophilic archaeon DL31] >gb|AEN06478.1|DEAD/DEAH box helicase domain protein [Halophilic archaeon DL31]XP_002716686.1 PREDICTED: DNA polymerase theta isoform 1 [Oryctolaguscuniculus] YP_656834.1 ATP-dependent DNA helicase [Haloquadratum walsbyiDSM 16790] >emb|CAJ51176.1| ATP-dependent DNA helicase [Haloquadratumwalsbyi DSM 16790] XP_003248103.1 PREDICTED: DNA polymerase theta-likeisoform 1 [Acyrthosiphon pisum] ABC72356.1 ATP-dependent DNA helicase[Haloquadratum walsbyi] CCC39031.1 DEAD/DEAH box helicase [Haloquadratumwalsbyi C23] XP_001165150.2 PREDICTED: DNA polymerase theta isoform 1[Pan troglodytes] XP_003225852.1 PREDICTED: DNA polymerase theta-like[Anolis carolinensis] XP_615375.3 PREDICTED: DNA polymerase theta [Bostaurus] >ref|XP_002684835.1| PREDICTED: polymerase (DNA directed),theta-like [Bos taurus] >gb|DAA33456.1| polymerase (DNA directed),theta-like [Bos taurus] XP_002813286.1 PREDICTED: LOW QUALITY PROTEIN:DNA polymerase theta-like [Pongo abelii] AAR08421.2 DNA polymerase theta[Homo sapiens] EAW79510.1 polymerase (DNA directed), theta, isoformCRA_a [Homo sapiens] NP_955452.3 DNA polymerase theta [Homosapiens] >sp|O75417.2|DPOLQ_HUMAN RecName: Full = DNA polymerase theta;AltName: Full = DNA polymerase eta >gb|AAI72289.1| Polymerase (DNAdirected), theta [synthetic polynucleotide] NP_001099348.1 DNApolymerase theta [Rattus norvegicus] >gb|EDM11249.1| polymerase (DNAdirected), theta (predicted), isoform CRA_a [Rattus norvegicus]XP_003341262.1 PREDICTED: LOW QUALITY PROTEIN: DNA polymerase theta-like[Monodelphis domestica] XP_001502374.3 PREDICTED: DNA polymerase theta[Equus caballus] XP_545125.3 PREDICTED: LOW QUALITY PROTEIN: DNApolymerase theta [Canis lupus familiaris] XP_002928855.1 PREDICTED: LOWQUALITY PROTEIN: DNA polymerase theta-like [Ailuropoda melanoleuca]NP_084253.1 DNA polymerase theta isoform 1 [Musmusculus] >gb|AAL77225.1| DNA polymerase theta [Musmusculus] >gb|EDK97951.1| polymerase (DNA directed), theta, isoformCRA_a [Mus musculus] >gb|AAI38361.1| Polymerase (DNA directed), theta[Mus musculus] >gb|AAI57901.1| Polymerase (DNA directed), theta [Musmusculus] AAK39635.1 DNA polymerase theta [Homo sapiens] AAN39838.1 DNApolymerase Q [Mus musculus] XP_003412882.1 PREDICTED: DNA polymerasetheta [Loxodonta africana] YP_003735206.1 DEAD/DEAH box helicasedomain-containing protein [Halalkalicoccus jeotgali B3] >gb|ADJ13414.1|DEAD/DEAH box helicase domain protein [Halalkalicoccus jeotgali B3]YP_004794841.1 pre-mRNA splicing helicase [Haloarcula hispanica ATCC33960] >gb|AEM55853.1| pre-mRNA splicing helicase [Haloarcula hispanicaATCC 33960] XP_416549.2 PREDICTED: similar to DNA polymerase theta[Gallus gallus] XP_003427319.1 PREDICTED: helicase POLQ-like isoform 2[Nasonia vitripennis] XP_003202748.1 PREDICTED: DNA polymerasetheta-like [Meleagris gallopavo] XP_969311.1 PREDICTED: similar to DNApolymerase theta [Tribolium castaneum] >gb|EEZ97532.1| hypotheticalprotein TcasGA2_TC011380 [Tribolium castaneum] ZP_08046037.1 DEAD/DEAHbox helicase domain protein [Haladaptatus paucihalophilusDX253] >gb|EFW90685.1| DEAD/DEAH box helicase domain protein[Haladaptatus paucihalophilus DX253] YP_461714.1 helicase [Syntrophusaciditrophicus SB] >gb|ABC77546.1| helicase [Syntrophus aciditrophicusSB] YP_003176510.1 DEAD/DEAH box helicase [Halomicrobium mukohataei DSM12286] >gb|ACV46803.1| DEAD/DEAH box helicase domain protein[Halomicrobium mukohataei DSM 12286] YP_137400.1 Pre-mRNA splicinghelicase [Haloarcula marismortui ATCC 43049] >gb|AAV47694.1| Pre-mRNAsplicing helicase [Haloarcula marismortui ATCC 43049] NP_001184156.1polymerase (DNA directed), theta [Xenopus (Silurana) tropicalis]NP_280861.1 pre-mRNA splicing helicase [Halobacterium sp.NRC-1] >ref|YP_001689987.1| ATP-dependent DNA helicase [Halobacteriumsalinarum R1] >gb|AAG20341.1| pre-mRNA splicing helicase [Halobacteriumsp. NRC-1] >emb|CAP14641.1| ATP-dependent DNA helicase [Halobacteriumsalinarum R1] YP_004595640.1 DEAD/DEAH box helicase domain-containingprotein [Halopiger xanaduensis SH-6] >gb|AEH35761.1| DEAD/DEAH boxhelicase domain protein [Halopiger xanaduensis SH-6] XP_001521144.2PREDICTED: DNA polymerase theta, partial [Ornithorhynchus anatinus]XP_003261953.1 PREDICTED: DNA polymerase theta, partial [Nomascusleucogenys] XP_001358456.2 GA19301 [Drosophila pseudoobscurapseudoobscura] >gb|EAL27595.2| GA19301 [Drosophila pseudoobscurapseudoobscura] ZP_08560003.1 DEAD/DEAH box helicase domain protein[Halorhabdus tiamatea SARL4B] >gb|EGM34502.1| DEAD/DEAH box helicasedomain protein [Halorhabdus tiamatea SARL4B] XP_002187783.1 PREDICTED:similar to polymerase (DNA directed), theta [Taeniopygia guttata]XP_002112587.1 hypothetical protein TRIADDRAFT_25163 [Trichoplaxadhaerens] >gb|EDV24697.1| hypothetical protein TRIADDRAFT_25163[Trichoplax adhaerens] YP_003405139.1 DEAD/DEAH box helicase[Haloterrigena turkmenica DSM 5511] >gb|ADB62466.1| DEAD/DEAH boxhelicase domain protein [Haloterrigena turkmenica DSM 5511] EGV92665.1DNA polymerase theta [Cricetulus griseus] CBY24305.1 unnamed proteinproduct [Oikopleura dioica] YP_003130565.1 DEAD/DEAH box helicase domainprotein [Halorhabdus utahensis DSM 12940] >gb|ACV11832.1| DEAD/DEAH boxhelicase domain protein [Halorhabdus utahensis DSM 12940] YP_003479811.1DEAD/DEAH box helicase [Natrialba magadii ATCC 43099] >gb|ADD05249.1|DEAD/DEAH box helicase domain protein [Natrialba magadii ATCC 43099]EFB22383.1 hypothetical protein PANDA_000253 [Ailuropoda melanoleuca]YP_003357334.1 putative ATP-dependent helicase [Methanocella paludicolaSANAE] >dbj|BAI62351.1| putative ATP-dependent helicase [Methanocellapaludicola SANAE] YP_325942.1 ATP-dependent DNA helicase 2 [Natronomonaspharaonis DSM 2160] >emb|CAI48373.2| ATP-dependent DNA helicase 2[Natronomonas pharaonis DSM 2160] XP_002912509.1 PREDICTED: LOW QUALITYPROTEIN: helicase POLQ-like [Ailuropoda melanoleuca] XP_002704678.1PREDICTED: helicase, POLQ-like [Bos taurus] CAE47762.2 novel proteinsimilar to humna DNA-directed polymerase theta (POLQ) [Danio rerio]XP_003205636.1 PREDICTED: helicase POLQ-like [Meleagris gallopavo]XP_544959.2 PREDICTED: helicase, POLQ-like [Canis lupus familiaris]EFX86757.1 hypothetical protein DAPPUDRAFT_312857 [Daphnia pulex]YP_003389641.1 DEAD/DEAH box helicase [Spirosoma linguale DSM74] >gb|ADB40842.1| DEAD/DEAH box helicase domain protein [Spirosomalinguale DSM 74] XP_002602932.1 hypothetical protein BRAFLDRAFT_251779[Branchiostoma floridae] >gb|EEN58944.1| hypothetical proteinBRAFLDRAFT_251779 [Branchiostoma floridae] YP_004144962.1 peptidase C14caspase catalytic subunit p20 [Mesorhizobium ciceri biovar biserrulaeWSM1271] >ref|YP_004614892.1| DEAD/DEAH box helicase domain-containingprotein [Mesorhizobium opportunistum WSM2075] >gb|ADV14912.1| peptidaseC14 caspase catalytic subunit p20 [Mesorhizobium ciceri biovarbiserrulae WSM1271] >gb|AEH90798.1| DEAD/DEAH box helicase domainprotein [Mesorhizobium opportunistum WSM2075] XP_002124758.1 PREDICTED:similar to DNA polymerase theta [Ciona intestinalis] XP_694437.5PREDICTED: DNA polymerase theta [Danio rerio] XP_420565.1 PREDICTED:similar to DNA helicase HEL308 [Gallus gallus] XP_003129397.1 PREDICTED:helicase POLQ-like [Sus scrofa] EDL20278.1 mCG128467, isoform CRA_b [Musmusculus] XP_001517710.2 PREDICTED: helicase POLQ, partial[Ornithorhynchus anatinus] AAH82601.1 Helicase, mus308-like (Drosophila)[Mus musculus] XP_003384429.1 PREDICTED: DNA polymerase theta-like[Amphimedon queenslandica] XP_003221282.1 PREDICTED: helicase POLQ-like[Anolis carolinensis] NP_524333.1 mutagen-sensitive 308 [Drosophilamelanogaster] >gb|AAB67306.1| Mus308 [Drosophilamelanogaster] >gb|AAF54858.1| mutagen-sensitive 308 [Drosophilamelanogaster] >gb|ACH92234.1| FI03732p [Drosophila melanogaster]AAX33507.1 LP14642p [Drosophila melanogaster] NP_001074576.1 helicasePOLQ-like [Mus musculus] >sp|Q2VPA6.2|HELQ_MOUSE RecName: Full =Helicase POLQ-like; AltName: Full = Mus308-like helicase; AltName: Full= POLQ-like helicase >gb|AAI09171.2| Helicase, mus308-like (Drosophila)[Mus musculus] YP_003523727.1 DEAD/DEAH box helicase domain protein[Sideroxydans lithotrophicus ES-1] >gb|ADE11340.1| DEAD/DEAH boxhelicase domain protein [Sideroxydans lithotrophicus ES-1]XP_002120889.1 PREDICTED: similar to DNA helicase HEL308 [Cionaintestinalis] XP_001892566.1 Type III restriction enzyme, res subunitfamily protein [Brugia malayi] >gb|EDP38603.1| Type III restrictionenzyme, res subunit family protein [Brugia malayi] ABZ09232.1 putativehelicase conserved C-terminal domain protein [uncultured marinecrenarchaeote HF4000_APKG7F11] XP_002814981.1 PREDICTED: LOW QUALITYPROTEIN: helicase POLQ-like [Pongo abelii] XP_002717082.1 PREDICTED: DNAhelicase HEL308 [Oryctolagus cuniculus] XP_001104832.1 PREDICTED:helicase, POLQ-like [Macaca mulatta] AAL85274.1 DNA helicase HEL308[Homo sapiens] NP_598375.2 helicase POLQ-like [Homosapiens] >gb|EAX05934.1| DNA helicase HEL308, isoform CRA_a [Homosapiens] >gb|AAI41525.1| Helicase, POLQ-like [synthetic polynucleotide]Q8TDG4.2 RecName: Full = Helicase POLQ-like; AltName: Full = Mus308-likehelicase; AltName: Full = POLQ-like helicase XP_003265889.1 PREDICTED:helicase POLQ [Nomascus leucogenys] XP_002745688.1 PREDICTED: helicasePOLQ-like [Callithrix jacchus] XP_003310356.1 PREDICTED: LOW QUALITYPROTEIN: helicase POLQ-like [Pan troglodytes] NP_001014156.2 helicase,POLQ-like [Rattus norvegicus] >ref|XP_001060858.1| PREDICTED: helicase,POLQ-like [Rattus norvegicus] >gb|EDL99554.1| rCG37823, isoform CRA_c[Rattus norvegicus] XP_001850567.1 ATP-dependent DNA helicase MER3[Culex quinquefasciatus] >gb|EDS32308.1| ATP-dependent DNA helicase MER3[Culex quinquefasciatus] XP_003427318.1 PREDICTED: helicase POLQ-likeisoform 1 [Nasonia vitripennis] XP_003143912.1 hypothetical proteinLOAG_08332 [Loa loa] >gb|EFO20157.1| hypothetical protein LOAG_08332[Loa loa] CAG11187.1 unnamed protein product [Tetraodon nigroviridis]XP_001111254.2 PREDICTED: DNA polymerase theta isoform 2 [Macacamulatta] XP_003414242.1 PREDICTED: helicase POLQ [Loxodonta africana]XP_002681870.1 predicted protein [Naegleria gruberi] >gb|EFC49126.1|predicted protein [Naegleria gruberi] EAX05935.1 DNA helicase HEL308,isoform CRA_b [Homo sapiens] AAH59917.1 Ascc3 protein [Mus musculus]ZP_07082808.1 DEAD/DEAH box helicase domain protein [Sphingobacteriumspiritivorum ATCC 33861] >gb|EFK55937.1| DEAD/DEAH box helicase domainprotein [Sphingobacterium spiritivorum ATCC 33861] XP_001494572.3PREDICTED: LOW QUALITY PROTEIN: helicase POLQ-like [Equus caballus]XP_002714920.1 PREDICTED: activating signal cointegrator 1 complexsubunit 3 [Oryctolagus cuniculus] XP_002598278.1 hypothetical proteinBRAFLDRAFT_204526 [Branchiostoma floridae] >gb|EEN54290.1| hypotheticalprotein BRAFLDRAFT_204526 [Branchiostoma floridae] XP_001943294.1PREDICTED: helicase POLQ-like isoform 1 [Acyrthosiphonpisum] >ref|XP_003240510.1| PREDICTED: helicase POLQ-like isoform 2[Acyrthosiphon pisum] XP_002803889.1 PREDICTED: activating signalcointegrator 1 complex subunit 3-like [Macaca mulatta] XP_001651546.1DNA polymerase theta [Aedes aegypti] >gb|EAT42599.1| DNA polymerasetheta [Aedes aegypti] CAA11679.1 RNA helicase [Homo sapiens]XP_002837795.1 hypothetical protein [Tuber melanosporumMel28] >emb|CAZ81986.1| unnamed protein product [Tuber melanosporum]EGT47882.1 hypothetical protein CAEBREN_02542 [Caenorhabditis brenneri]EDL99655.1 activating signal cointegrator 1 complex subunit 3(predicted), isoform CRA_b [Rattus norvegicus] NP_932124.2 activatingsignal cointegrator 1 complex subunit 3 [Mus musculus] EDL05054.1mCG119534 [Mus musculus] gi|352115865 DEAD/DEAH box helicase domainprotein ZP_08963952.1 [Natrinema pellirubrum DSM 15624]

The Hel308 helicase is more preferably one of the helicases shown inTable 5 below or a variant thereof. The Hel308 helicase more preferablycomprises the sequence of one of the helicases shown in Table 5, i.e.one of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and58, or a variant thereof.

TABLE 5  More preferred Hel308 helicases and most preferred Hel308motifs and extended Hel308 motifs % % SEQ Identity Identity ID Hel308Hel308 Hel308 Extended Hel308 NO: Helicase Names Pfu Mbu motif motif 10Hel308 Mbu Methanococcoides   37% — QMAGRAGR QMAGRAGRP burtonii(SEQ ID NO: (SEQ ID NO: 12) 11) 13 Hel308 Pfu Pyrococcus — 37% QMLGRAGRQMLGRAGRP furiosus DSM (SEQ ID NO: (SEQ ID NO: 15) 3638 14) 16Hel308 Hvo Haloferax    34% 41% QMMGRAGR QMMGRAGRP volcanii (SEQ ID NO:(SEQ ID NO: 18) 17) 19 Hel308 Hla Halorubrum   35% 42% QMCGRAGRQMCGRAGRP lacusprofundi (SEQ ID NO: (SEQ ID NO: 21) 20) 22 Hel308 CsyCenarchaeum   34% 34% QLCGRAGR QLCGRAGRP symbiosum (SEQ ID NO:(SEQ ID NO: 24) 23) 25 Hel308 Sso Sulfolobus   35% 33% QMSGRAGRQMSGRAGRP solfataricus (SEQ ID NO: (SEQ ID NO: 27) 26) 28 Hel308 MfrMethanogenium   37% 44% QMAGRAGR QMAGRAGRP frigidum (SEQ ID NO:(SEQ ID NO: 12) 11) 29 Hel308 Mok Methanothermococcus   37% 34% QCIGRAGRQCIGRAGRP okinawensis (SEQ ID NO: (SEQ ID NO: 31) 30) 32 Hel308 MigMethanotorris   40% 35% QCIGRAGR QCIGRAGRP igneus Kol 5 (SEQ ID NO:(SEQ ID NO: 31) 30) 33 Hel308 Tga Thermococcus   60% 38% QMMGRAGRQMMGRAGRP gammatolerans (SEQ ID NO: (SEQ ID NO: 18) EJ3 17) 34Hel308 Tba Thermococcus   57% 35% QMIGRAGR QMIGRAGRP barophilus MP(SEQ ID NO: (SEQ ID NO: 36) 35) 37 Hel308 Tsi Thermococcus   56% 35%QMMGRAGR QMMGRAGRP sibmcus MM 739 (SEQ ID NO: (SEQ ID NO: 18) 17) 38Hel308 Mba Methanosarcina   39% 60% QMAGRAGR QMAGRAGRP barkeri str. (SEQ ID NO: (SEQ ID NO: 12) Fusaro 11) 39 Hel308 Mac Methanosarcina  38% 60% QMAGRAGR QMAGRAGRP acetivorans (SEQ ID NO: (SEQ ID NO: 12) 11)40 Hel308 Methanohalophilus   38% 60% QMAGRAGR QMAGRAGRP Mmahmahii DSM 5219 (SEQ ID NO: (SEQ ID NO: 12) 11) 41 Hel308 Methanosarcina  38% 60% QMAGRAGR QMAGRAGRP Mmaz mazei (SEQ ID NO: (SEQ ID NO: 12) 11)42 Hel308 Mth Methanosaeta   39% 46% QMAGRAGR QMAGRAGRP thermophila PT(SEQ ID NO: (SEQ ID NO: 12) 11) 43 Hel308 Mzh Methanosalsum   39% 57%QMAGRAGR QMAGRAGRP zhilinae DSM (SEQ ID NO: (SEQ ID NO: 12) 4017 11) 44Hel308 Mev Methanohalobium   38% 61% QMAGRAGR QMAGRAGRP evestigatum Z-(SEQ ID NO: (SEQ ID NO: 12) 7303 11) 45 Hel308 Mma Methanococcus   36%32% QCIGRAGR QCIGRAGRP maripaludis (SEQ ID NO: (SEQ ID NO: 31) 30) 46Hel308 Nma Natrialba magadii   37% 43% QMMGRAGR QMMGRAGRP (SEQ ID NO:(SEQ ID NO: 18) 17) 47 Hel308 Mbo Methanoregula   38% 45% QMAGRAGRQMAGRAGRP boonei 6A8 (SEQ ID NO: (SEQ ID NO: 12) 11) 48 Hel308 FacFerroplasma   34% 32% QMIGRAGR QMIGRAGRP acidarmanus (SEQ ID NO:(SEQ ID NO: 36) 35) 49 Hel308 Mfe Methanocaldococcus   40% 35% QCIGRAGRQCIGRAGRP fervens AG86 (SEQ ID NO: (SEQ ID NO: 31) 30) 50 Hel308 MjaMethanocaldococcus   24% 22% QCIGRAGR QCIGRAGRP jannaschii (SEQ ID NO:(SEQ ID NO: 31) 30) 51 Hel308 Mm Methanocaldococcus   41% 33% QCIGRAGRQCIGRAGRP infernus (SEQ ID NO: (SEQ ID NO: 31) 30) 52 Hel308 MhuMethanospirillum   36% 40% QMAGRAGR QMAGRAGRP hungatei JF-1 (SEQ ID NO:(SEQ ID NO: 12) 11) 53 Hel308 Afu Archaeoglobus   40% 40% QMAGRAGRQMAGRAGRP fulgidus DSM (SEQ ID NO: (SEQ ID NO: 12) 4304 11) 54Hel308 Htu Haloterrigena   35% 43% QMAGRAGR QMMGRAGRP turkmenica(SEQ ID NO: (SEQ ID NO: 12) 11) 55 Hel308 Hpa Haladaptatus   38% 45%QMFGRAGR QMFGRAGRP DX253 paucihalophilus (SEQ ID NO: (SEQ ID NO: 57) 56)58 ski2-like Halobacterium sp. 36.8% 42.0% QMFGRAGR QMFGRAGRP helicaseNRC-1 (SEQ ID NO: (SEQ ID NO: 57) 56)

The Hel308 helicase more preferably comprises (a) the sequence of Hel308Mbu (i.e. SEQ ID NO: 10) or a variant thereof, (b) the sequence ofHel308 Pfu (i.e. SEQ ID NO: 13) or a variant thereof, (c) the sequenceof Hel308 Mok (i.e. SEQ ID NO: 29) or a variant thereof, (d) thesequence of Hel308 Mma (i.e. SEQ ID NO: 45) or a variant thereof, (e)the sequence of Hel308 Fac (i.e. SEQ ID NO: 48) or a variant thereof or(f) the sequence of Hel308 Mhu (i.e. SEQ ID NO: 52) or a variantthereof. The Hel308 helicase more preferably comprises the sequenceshown in SEQ ID NO: 10 or a variant thereof.

The Hel308 helicase more preferably comprises (a) the sequence of Hel308Tga (i.e. SEQ ID NO: 33) or a variant thereof, (b) the sequence ofHel308 Csy (i.e. SEQ ID NO: 22) or a variant thereof or (c) the sequenceof Hel308 Mhu (i.e. SEQ ID NO: 52) or a variant thereof. The Hel308helicase most preferably comprises the sequence shown in SEQ ID NO: 33or a variant thereof.

A variant of a Hel308 helicase is an enzyme that has an amino acidsequence which varies from that of the wild-type helicase and whichretains polynucleotide binding activity. In particular, a variant of anyone of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and58 is an enzyme that has an amino acid sequence which varies from thatof any one of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55 and 58 and which retains polynucleotide binding activity. A variantof SEQ ID NO: 10 or 33 is an enzyme that has an amino acid sequencewhich varies from that of SEQ ID NO: 10 or 33 and which retainspolynucleotide binding activity. The variant retains helicase activity.The variant must work in at least one of the two modes discussed below.Preferably, the variant works in both modes. The variant may includemodifications that facilitate handling of the polynucleotide encodingthe helicase and/or facilitate its activity at high salt concentrationsand/or room temperature. Variants typically differ from the wild-typehelicase in regions outside of the Hel308 motif or extended Hel308 motifdiscussed above. However, variants may include modifications withinthese motif(s).

Over the entire length of the amino acid sequence of any one of SEQ IDNOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58, such as SEQID NO: 10 or 33, a variant will preferably be at least 30% homologous tothat sequence based on amino acid identity. More preferably, the variantpolypeptide may be at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90% and more preferably at least 95%, 97% or99% homologous based on amino acid identity to the amino acid sequenceof any one of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55 and 58, such as SEQ ID NO: 10 or 33, over the entire sequence. Theremay be at least 70%, for example at least 80%, at least 85%, at least90% or at least 95%, amino acid identity over a stretch of 150 or more,for example 200, 300, 400, 500, 600, 700, 800, 900 or 1000 or more,contiguous amino acids (“hard homology”). Homology is determined asdescribed above. The variant may differ from the wild-type sequence inany of the ways discussed above with reference to SEQ ID NOs: 2 and 4.

A variant of any one of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32,33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55 and 58 preferably comprises the Hel308 motif or extendedHel308 motif of the relevant wild-type sequence. For instance, a variantof SEQ ID NO: 10 preferably comprises the Hel308 motif of SEQ ID NO: 10(QMAGRAGR; SEQ ID NO: 11) or extended Hel308 motif of SEQ ID NO: 10(QMAGRAGRP; SEQ ID NO: 12). The Hel308 motif and extended Hel308 motifof each of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55and 58 are shown in Table 5. However, a variant of any one SEQ ID NOs:10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and 58 may comprise theHel308 motif or extended Hel308 motif from a different wild-typesequence. For instance, a variant of SEQ ID NO: 10 may comprise theHel308 motif of SEQ ID NO: 13 (QMLGRAGR; SEQ ID NO: 14) or extendedHel308 motif of SEQ ID NO: 13 (QMLGRAGRP; SEQ ID NO: 15). A variant ofany one SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 and58 may comprise any one of the preferred motifs shown in Table 5.Variants of any one of SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32,33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55 and 58 may also include modifications within the Hel308 motifor extended Hel308 motif of the relevant wild-type sequence. Suitablemodifications at X1 and X2 are discussed above when defining the twomotifs.

A variant of SEQ ID NO: 10 may lack the first 19 amino acids of SEQ IDNO: 10 and/or lack the last 33 amino acids of SEQ ID NO: 10. A variantof SEQ ID NO: 10 preferably comprises a sequence which is at least 70%,at least 75%, at least 80%, at least 85%, at least 90% or morepreferably at least 95%, at least 97% or at least 99% homologous basedon amino acid identity with amino acids 20 to 211 or 20 to 727 of SEQ IDNO: 10.

The helicase may be covalently attached to the pore. The helicase ispreferably not covalently attached to the pore. The application of avoltage to the pore and helicase typically results in the formation of asensor that is capable of sequencing target polynucleotides. This isdiscussed in more detail below.

Any of the proteins described herein, i.e. the transmembrane proteinpores or Hel308 helicases, may be modified to assist theiridentification or purification, for example by the addition of histidineresidues (a his tag), aspartic acid residues (an asp tag), astreptavidin tag, a flag tag, a SUMO tag, a GST tag or a MBP tag, or bythe addition of a signal sequence to promote their secretion from a cellwhere the polypeptide does not naturally contain such a sequence. Analternative to introducing a genetic tag is to chemically react a tagonto a native or engineered position on the pore or helicase. An exampleof this would be to react a gel-shift reagent to a cysteine engineeredon the outside of the pore. This has been demonstrated as a method forseparating hemolysin hetero-oligomers (Chem Biol. 1997 July;4(7):497-505).

The pore and/or helicase may be labelled with a revealing label. Therevealing label may be any suitable label which allows the pore to bedetected. Suitable labels include, but are not limited to, fluorescentmolecules, radioisotopes, e.g. ¹²⁵I ³⁵S, enzymes, antibodies, antigens,polynucleotides and ligands such as biotin.

Proteins may be made synthetically or by recombinant means. For example,the pore and/or helicase may be synthesized by in vitro translation andtranscription (IVTT). The amino acid sequence of the pore and/orhelicase may be modified to include non-naturally occurring amino acidsor to increase the stability of the protein. When a protein is producedby synthetic means, such amino acids may be introduced duringproduction. The pore and/or helicase may also be altered followingeither synthetic or recombinant production.

The pore and/or helicase may also be produced using D-amino acids. Forinstance, the pore or helicase may comprise a mixture of L-amino acidsand D-amino acids. This is conventional in the art for producing suchproteins or peptides.

The pore and/or helicase may also contain other non-specificmodifications as long as they do not interfere with pore formation orhelicase function. A number of non-specific side chain modifications areknown in the art and may be made to the side chains of the protein(s).Such modifications include, for example, reductive alkylation of aminoacids by reaction with an aldehyde followed by reduction with NaBH₄,amidination with methylacetimidate or acylation with acetic anhydride.

The pore and helicase can be produced using standard methods known inthe art. Polynucleotide sequences encoding a pore or helicase may bederived and replicated using standard methods in the art. Polynucleotidesequences encoding a pore or helicase may be expressed in a bacterialhost cell using standard techniques in the art. The pore and/or helicasemay be produced in a cell by in situ expression of the polypeptide froma recombinant expression vector. The expression vector optionallycarries an inducible promoter to control the expression of thepolypeptide. These methods are described in described in Sambrook, J.and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rdEdition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

The pore and/or helicase may be produced in large scale followingpurification by any protein liquid chromatography system from proteinproducing organisms or after recombinant expression. Typical proteinliquid chromatography systems include FPLC, AKTA systems, the Bio-Cadsystem, the Bio-Rad BioLogic system and the Gilson HPLC system.

The method of the invention involves measuring one or morecharacteristics of the target polynucleotide. The method may involvemeasuring two, three, four or five or more characteristics of the targetpolynucleotide. The one or more characteristics are preferably selectedfrom (i) the length of the target polynucleotide, (ii) the identity ofthe target polynucleotide, (iii) the sequence of the targetpolynucleotide, (iv) the secondary structure of the targetpolynucleotide and (v) whether or not the target polynucleotide ismodified. Any combination of (i) to (v) may be measured in accordancewith the invention.

For (i), the length of the polynucleotide may be measured using thenumber of interactions between the target polynucleotide and the pore.

For (ii), the identity of the polynucleotide may be measured in a numberof ways. The identity of the polynucleotide may be measured inconjunction with measurement of the sequence of the targetpolynucleotide or without measurement of the sequence of the targetpolynucleotide. The former is straightforward; the polynucleotide issequenced and thereby identified. The latter may be done in severalways. For instance, the presence of a particular motif in thepolynucleotide may be measured (without measuring the remaining sequenceof the polynucleotide). Alternatively, the measurement of a particularelectrical and/or optical signal in the method may identify the targetpolynucleotide as coming from a particular source.

For (iii), the sequence of the polynucleotide can be determined asdescribed previously. Suitable sequencing methods, particularly thoseusing electrical measurements, are described in Stoddart D et al., ProcNatl Acad Sci, 12; 106(19):7702-7, Lieberman K R et al, J Am Chem Soc.2010; 132(50):17961-72, and International Application WO 2000/28312.

For (iv), the secondary structure may be measured in a variety of ways.For instance, if the method involves an electrical measurement, thesecondary structure may be measured using a change in dwell time or achange in current flowing through the pore. This allows regions ofsingle-stranded and double-stranded polynucleotide to be distinguished.

For (v), the presence or absence of any modification may be measured.The method preferably comprises determining whether or not the targetpolynucleotide is modified by methylation, by oxidation, by damage, withone or more proteins or with one or more labels, tags or spacers.Specific modifications will result in specific interactions with thepore which can be measured using the methods described below. Forinstance, methylcyotsine may be distinguished from cytosine on the basisof the current flowing through the pore during its interation with eachnucleotide.

A variety of different types of measurements may be made. This includeswithout limitation: electrical measurements and optical measurements.Possible electrical measurements include: current measurements,impedance measurements, tunnelling measurements (Ivanov A P et al., NanoLett. 2011 Jan. 12; 11(1):279-85), and FET measurements (InternationalApplication WO 2005/124888). Optical measurements may be combined 10with electrical measurements (Soni G V et al., Rev Sci Instrum. 2010January; 81(1):014301). The measurement may be a transmembrane currentmeasurement such as measurement of ionic current flowing through thepore.

Electrical measurements may be made using standard single channelrecording equipment as describe in Stoddart D et al., Proc Natl AcadSci, 12; 106(19):7702-7, Lieberman K R et al, J Am Chem Soc. 2010;132(50):17961-72, and International Application WO-2000/28312.Alternatively, electrical measurements may be made using a multi-channelsystem, for example as described in International ApplicationWO-2009/077734 and International Application WO-2011/067559.

In a preferred embodiment, the method comprises:

(a) contacting the target polynucleotide with a transmembrane pore and aHel308 helicase such that the helicase controls the movement of thetarget polynucleotide through the pore and nucleotides in the targetpolynucleotide interact with the pore; and

(b) measuring the current passing through the pore during one or moreinteractions to measure one or more characteristics of the targetpolynucleotide and thereby characterising the target polynucleotide.

The methods may be carried out using any apparatus that is suitable forinvestigating a membrane/pore system in which a pore is inserted into amembrane. The method may be carried out using any apparatus that issuitable for transmembrane pore sensing. For example, the apparatuscomprises a chamber comprising an aqueous solution and a barrier thatseparates the chamber into two sections. The barrier has an aperture inwhich the membrane containing the pore is formed.

The methods may be carried out using the apparatus described inInternational Application No. PCT/GB08/000562 (WO 2008/102120).

The methods may involve measuring the current passing through the poreduring one or more interactions with the nucleotide(s). Therefore theapparatus may also comprise an electrical circuit capable of applying apotential and measuring an electrical signal across the membrane andpore. The methods may be carried out using a patch clamp or a voltageclamp. The methods preferably involve the use of a voltage clamp.

The methods of the invention may involve the measuring of a currentpassing through the pore during one or more interactions with thenucleotide. Suitable conditions for measuring ionic currents throughtransmembrane protein pores are known in the art and disclosed in theExample. The method is typically carried out with a voltage appliedacross the membrane and pore. The voltage used is typically from +2 V to−2 V, typically −400 mV to +400 mV. The voltage used is preferably in arange having a lower limit selected from −400 mV, −300 mV, −200 mV, −150mV, −100 mV, −50 mV, −20 mV and 0 mV and an upper limit independentlyselected from +10 mV, +20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mVand +400 mV. The voltage used is more preferably in the range 100 mV to240 mV and most preferably in the range of 120 mV to 220 mV. It ispossible to increase discrimination between different nucleotides by apore by using an increased applied potential.

The methods are typically carried out in the presence of any chargecarriers, such as metal salts, for example alkali metal salt, halidesalts, for example chloride salts, such as alkali metal chloride salt.Charge carriers may include ionic liquids or organic salts, for exampletetramethyl ammonium chloride, trimethylphenyl ammonium chloride,phenyltrimethyl ammonium chloride, or 1-ethyl-3-methyl imidazoliumchloride. In the exemplary apparatus discussed above, the salt ispresent in the aqueous solution in the chamber. Potassium chloride(KCl), sodium chloride (NaCl) or caesium chloride (CsCl) is typicallyused. KCl is preferred. The salt concentration may be at saturation. Thesalt concentration may be 3M or lower and is typically from 0.1 to 2.5M, from 0.3 to 1.9 M, from 0.5 to 1.8 M, from 0.7 to 1.7 M, from 0.9 to1.6 M or from 1 M to 1.4 M. The salt concentration is preferably from150 mM to 1 M. As discussed above, Hel308 helicases surprisingly workunder high salt concentrations. The method is preferably carried outusing a salt concentration of at least 0.3 M, such as at least 0.4 M, atleast 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least1.5 M, at least 2.0 M, at least 2.5 M or at least 3.0 M. High saltconcentrations provide a high signal to noise ratio and allow forcurrents indicative of the presence of a nucleotide to be identifiedagainst the background of normal current fluctuations.

The methods are typically carried out in the presence of a buffer. Inthe exemplary apparatus discussed above, the buffer is present in theaqueous solution in the chamber. Any buffer may be used in the method ofthe invention. Typically, the buffer is HEPES. Another suitable bufferis Tris-HCl buffer. The methods are typically carried out at a pH offrom 4.0 to 12.0, from 4.5 to 10.0, from 5.0 to 9.0, from 5.5 to 8.8,from 6.0 to 8.7 or from 7.0 to 8.8 or 7.5 to 8.5. The pH used ispreferably about 7.5.

The methods may be carried out at from 0° C. to 100° C., from 15° C. to95° C., from 16° C. to 90° C., from 17° C. to 85° C., from 18° C. to 80°C., 19° C. to 70° C., or from 20° C. to 60° C. The methods are typicallycarried out at room temperature. The methods are optionally carried outat a temperature that supports enzyme function, such as about 37° C.

The method is typically carried out in the presence of free nucleotidesor free nucleotide analogues and an enzyme cofactor that facilitate theaction of the helicase. The free nucleotides may be one or more of anyof the individual nucleotides discussed above. The free nucleotidesinclude, but are not limited to, adenosine monophosphate (AMP),adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosinemonophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate(GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP),thymidine triphosphate (TTP), uridine monophosphate (UMP), uridinediphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate(CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclicadenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP),deoxyadenosine monophosphate (dAMP), deoxy adenosine diphosphate (dADP),deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP),deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP),deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP),deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP),deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP),deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) anddeoxycytidine triphosphate (dCTP). The free nucleotides are preferablyselected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP. Thefree nucleotides are preferably adenosine triphosphate (ATP). The enzymecofactor is a factor that allows the helicase to function. The enzymecofactor is preferably a divalent metal cation. The divalent metalcation is preferably Mg²⁺, Mn²⁺, Ca²⁺ or Co²⁺. The enzyme cofactor ismost preferably Mg²⁺.

The target polynucleotide may be contacted with the Hel308 helicase andthe pore in any order. In is preferred that, when the targetpolynucleotide is contacted with the Hel308 helicase and the pore, thetarget polynucleotide firstly forms a complex with the helicase. Whenthe voltage is applied across the pore, the targetpolynucleotide/helicase complex then forms a complex with the pore andcontrols the movement of the polynucleotide through the pore.

As discussed above, Hel308 helicases may work in two modes with respectto the nanopore. First, the method is preferably carried out using theHel308 helicase such that it moves the target sequence through the porewith the field resulting from the applied voltage. In this mode the 3′end of the DNA is first captured in the nanopore, and the enzyme movesthe DNA into the nanopore such that the target sequence is passedthrough the nanopore with the field until it finally translocatesthrough to the trans side of the bilayer. Alternatively, the method ispreferably carried out such that the enzyme moves the target sequencethrough the pore against the field resulting from the applied voltage.In this mode the 5′ end of the DNA is first captured in the nanopore,and the enzyme moves the DNA through the nanopore such that the targetsequence is pulled out of the nanopore against the applied field untilfinally ejected back to the cis side of the bilayer.

The method of the invention most preferably involves a pore derived fromMspA and a helicase comprising the sequence shown in SEQ ID NO: 8 or 10or a variant thereof. Any of the embodiments discussed above withreference to MspA and SEQ ID NO: 8 and 10 may be used in combination.

Other Methods

The invention also provides a method of forming a sensor forcharacterising a target polynucleotide. The method comprises forming acomplex between a pore and a Hel308 helicase. The complex may be formedby contacting the pore and the helicase in the presence of the targetpolynucleotide and then applying a potential across the pore. Theapplied potential may be a chemical potential or a voltage potential asdescribed above. Alternatively, the complex may be formed by covalentlyattaching the pore to the helicase. Methods for covalent attachment areknown in the art and disclosed, for example, in InternationalApplication Nos. PCT/GB09/001679 (published as WO 2010/004265) andPCT/GB10/000133 (published as WO 2010/086603). The complex is a sensorfor characterising the target polynucleotide. The method preferablycomprises forming a complex between a pore derived from Msp and a Hel308helicase. Any of the embodiments discussed above with reference to themethod of the invention equally apply to this method.

Kits

The present invention also provides kits for characterising a targetpolynucleotide. The kits comprise (a) a pore and (b) a Hel308 helicase.Any of the embodiments discussed above with reference to the method ofthe invention equally apply to the kits.

The kit may further comprise the components of a membrane, such as thephospholipids needed to form a lipid bilayer.

The kits of the invention may additionally comprise one or more otherreagents or instruments which enable any of the embodiments mentionedabove to be carried out. Such reagents or instruments include one ormore of the following: suitable buffer(s) (aqueous solutions), means toobtain a sample from a subject (such as a vessel or an instrumentcomprising a needle), means to amplify and/or express polynucleotides, amembrane as defined above or voltage or patch clamp apparatus. Reagentsmay be present in the kit in a dry state such that a fluid sampleresuspends the reagents. The kit may also, optionally, compriseinstructions to enable the kit to be used in the method of the inventionor details regarding which patients the method may be used for. The kitmay, optionally, comprise nucleotides.

Apparatus

The invention also provides an apparatus for characterising a targetpolynucleotide. The apparatus comprises a plurality of pores and aplurality of a Hel308 helicase. The apparatus preferably furthercomprises instructions for carrying out the method of the invention. Theapparatus may be any conventional apparatus for polynucleotide analysis,such as an array or a chip. Any of the embodiments discussed above withreference to the methods of the invention are equally applicable to theapparatus of the invention.

The apparatus is preferably set up to carry out the method of theinvention.

The Apparatus Preferably Comprises:

a sensor device that is capable of supporting the membrane and pluralityof pores and being operable to perform polynucleotide characterisingusing the pores and helicases;

at least one reservoir for holding material for performing thecharacterising;

a fluidics system configured to controllably supply material from the atleast one reservoir to the sensor device; and

a plurality of containers for receiving respective samples, the fluidicssystem being configured to supply the samples selectively from thecontainers to the sensor device. The apparatus may be any of thosedescribed in International Application No. PCT/GB08/004127 (published asWO 2009/077734), PCT/GB10/000789 (published as WO 2010/122293),International Application No. PCT/GB10/002206 (not yet published) orInternational Application No. PCT/US99/25679 (published as WO 00/28312).

Internally Binding Molecular Motors

Molecular motors are commonly used as a means for controlling thetranslocation of a polymer, particularly a polynucleotide, through ananopore. Surprisingly, the inventors have found that molecular motorswhich are capable of binding to a target polynucleotide at an internalnucleotide, i.e. a position other than a 5′ or 3′ terminal nucleotide,can provide increased read lengths of the polynucleotide as themolecular motor controls the translocation of the polynucleotide througha nanopore. The ability to translocate an entire polynucleotide througha nanopore under the control of a molecular motor allows characteristicsof the polynucleotide, such as its sequence, to be estimated withimproved accuracy and speed over known methods. This becomes moreimportant as strand lengths increase and molecular motors are requiredwith improved processivity. The molecular motor used in the invention isparticularly effective in controlling the translocation of targetpolynucleotides of 500 nucleotides or more, for example 1000nucleotides, 5000, 10000 or 20000 or more.

The invention thus provides a method of characterising a targetpolynucleotide, comprising:

(a) contacting the target polynucleotide with a transmembrane pore and amolecular motor which is capable of binding to the target polynucleotideat an internal nucleotide such that the molecular motor controls themovement of the target polynucleotide through the pore and nucleotidesin the target polynucleotide interact with the pore; and

(b) measuring one or more characteristics of the target polynucleotideduring one or more interactions and thereby characterising the targetpolynucleotide.

Any of the embodiments discussed above in relation to the Hel308 methodsof the invention equally apply to this method of the invention.

A problem which occurs in sequencing polynucleotides, particularly thoseof 500 nucleotides or more, is that the molecular motor which iscontrolling translocation of the polynucleotide may disengage from thepolynucleotide. This allows the polynucleotide to be pulled through thepore rapidly and in an uncontrolled manner in the direction of theapplied field. Multiple instances of the molecular motor used in theinvention bind to the polynucleotide at relatively short distances apartand thus the length of polynucleotide which can be pulled through thepore before a further molecular motor engages with the pore isrelatively short.

An internal nucleotide is a nucleotide which is not a terminalnucleotide in the target polynucleotide. For example, it is not a 3′terminal nucleotide or a 5′ terminal nucleotide. All nucleotides in acircular polynucleotide are internal nucleotides.

Generally, a molecular motor which is capable of binding at an internalnucleotide is also capable of binding at a terminal nucleotide, but thetendency for some molecular motors to bind at an internal nucleotidewill be greater than others. For a molecular motor suitable for use inthe invention, typically at least 10% of its binding to a polynucleotidewill be at an internal nucleotide. Typically, at least 20%, at least30%, at least 40% or at least 50% of its binding will be at an internalnucleotide. Binding at a terminal nucleotide may involve binding to botha terminal nucleotide and adjacent internal nucleotides at the sametime. For the purposes of the invention, this is not binding to thetarget polynucleotide at an internal nucleotide. In other words, themolecular motor used in the invention is not only capable of binding toa terminal nucleotide in combination with one or more adjacent internalnucleotides. The molecular motor must be capable of binding to aninternal nucleotide without concurrent binding to a terminal nucleotide.

A molecular motor which is capable of binding at an internal nucleotidemay bind to more than one internal nucleotide. Typically, the molecularmotor binds to at least 2 internal nucleotides, for example at least 3,at least 4, at least 5, at least 10 or at least 15 internal nucleotides.Typically the molecular motor binds to at least 2 adjacent internalnucleotides, for example at least 3, at least 4, at least 5, at least 10or at least 15 adjacent internal nucleotides. The at least 2 internalnucleotides may be adjacent or non-adjacent.

The ability of a molecular motor to bind to a polynucleotide at aninternal nucleotide may be determined by carrying out a comparativeassay. The ability of a motor to bind to a control polynucleotide A iscompared to the ability to bind to the same polynucleotide but with ablocking group attached at the terminal nucleotide (polynucleotide B).The blocking group prevents any binding at the terminal nucleotide ofstrand B, and thus allows only internal binding of a molecular motor. Anexample of this type of assay is disclosed in Example 4.

Suitable molecular motors are well known in the art and typicallyinclude, but are not limited to, single and double strand translocases,such as polymerases, helicases, topoisomerases, ligases and nucleases,such as exonucleases. Preferably the molecular motor is a helicase, forexample a Hel308 helicase. Examples of Hel308 helicases which arecapable of binding at an internal nucleotide include, but are notlimited to, Hel308 Tga, Hel308 Mhu and Hel308 Csy. Hence, the molecularmotor preferably comprises (a) the sequence of Hel308 Tga (i.e. SEQ IDNO: 33) or a variant thereof or (b) the sequence of Hel308 Csy (i.e. SEQID NO: 22) or a variant thereof or (c) the sequence of Hel308 Mhu (i.e.SEQ ID NO: 52) or a variant thereof. The variant typically has at least40% homology to SEQ ID NO: 33, 22 or 52 based on amino acid identityover the entire sequence and retains helicase activity. Further possiblevariants are discussed above.

The molecular motor used in the invention may be made by any of themethods discussed above and may be modified or labelled as discussedabove. The molecular motor may be used in the methods described hereinor as part of the apparatus described herein. The invention furtherprovides a method of forming a sensor for characterising a targetpolynucleotide, comprising forming a complex between a pore and amolecular motor which is capable of binding to the target polynucleotideat an internal nucleotide and thereby forming a sensor forcharacterising the target polynucleotide. The invention also providesuse of a molecular motor which is capable of binding to the targetpolynucleotide at an internal nucleotide to control the movement of atarget polynucleotide through a pore. The invention also provides a kitfor characterising a target polynucleotide comprising (a) a pore and (b)a molecular motor which is capable of binding to the targetpolynucleotide at an internal nucleotide. The invention also provides ananalysis apparatus for characterising target polynucleotides in asample, comprising a plurality of pores and a plurality of a molecularmotor which is capable of binding to the target polynucleotide at aninternal nucleotide.

The following Examples illustrate the invention.

Example 1

This Example illustrates the use of a Hel308 helicase (Hel308 MBu) tocontrol the movement of intact DNA strands through a nanopore. Thegeneral method and substrate employed throughout this example is shownin FIGS. 1A-1B and described in the figure caption.

Materials and Methods

Primers were designed to amplify a ˜400 bp fragment of PhiX174. Each ofthe 5′-ends of these primers included a 50 nucleotide non-complimentaryregion, either a homopolymeric stretch or repeating units of 10nucleotide homopolymeric sections. These serve as identifiers forcontrolled translocation of the strand through a nanopore, as well asdetermining the directionality of translocation. In addition, the 5′-endof the forward primer was “capped” to include four 2′-O-Methyl-Uracil(mU) nucleotides and the 5′-end of the reverse primer was chemicallyphosphorylated. These primer modifications then allow for the controlleddigestion of predominantly only the antisense strand, using lambdaexonuclease. The mU capping protects the sense strand from nucleasedigestion whilst the PO4 at the 5′ of the antisense strand promotes it.Therefore after incubation with lambda exonuclease only the sense strandof the duplex remains intact, now as single stranded DNA (ssDNA). Thegenerated ssDNA was then PAGE purified as previously described.

The DNA substrate design used in all the experiments described here isshown in FIG. 6A. The DNA substrate consists of a 400base section ofssDNA from PhiX, with a 50T 5′-leader to aid capture by the nanopore(SEQ ID NO: 59). Annealed to this strand just after the 50T leader is aprimer (SEQ ID NO: 60) containing a 3′ cholesterol tag to enrich the DNAon the surface of the bilayer, and thus improve capture efficiency.

Buffered solution: 400 mM-2 M KCl, 10 mM Hepes pH 8.0, 1 mM ATP, 1 mMMgCl₂, 1 mM DTT

Nanopore: E. coli MS(B2)8 MspA ONLP3271 MS-(L88N/D90N/D91N/D93N/10D118R/D134R/E139K)8

Enzyme: Hel308 Mbu (ONLP3302, ˜7.7 μM) 12.5 μl→100 nM final.

Electrical measurements were acquired from single MspA nanoporesinserted in 1,2-diphytanoyl-glycero-3-phosphocholine lipid (Avanti PolarLipids) bilayers. Bilayers were formed across ˜100 μm diameter aperturesin 20 μm thick PTFE films (in custom Delrin chambers) via theMontal-Mueller technique, separating two 1 mL buffered solutions. Allexperiments were carried out in the stated buffered solution.Single-channel currents were measured on Axopatch 200B amplifiers(Molecular Devices) equipped with 1440A digitizers. Ag/AgCl electrodeswere connected to the buffered solutions so that the cis compartment (towhich both nanopore and enzyme/DNA are added) is connected to the groundof the Axopatch headstage, and the trans compartment is connected to theactive electrode of the headstage. After achieving a single pore in thebilayer, DNA polynucleotide and helicase were added to 100 μL of bufferand pre-incubated for 5 mins (DNA=1.5 nM, Enzyme=1 μM). Thispre-incubation mix was added to 900 μL of buffer in the cis compartmentof the electrophysiology chamber to initiate capture of the helicase-DNAcomplexes in the MspA nanopore (to give final concentrations of DNA=0.15nM, Enzyme=0.1 μM). Helicase ATPase activity was initiated as requiredby the addition of divalent metal (1 mM MgCl₂) and NTP (1 mM ATP) to thecis compartment. Experiments were carried out at a constant potential of+180 mV.

Results and Discussion

The addition of Helicase-DNA substrate to MspA nanopores as shown inFIGS. 1A-1B produces characteristic current blocks as shown in FIG. 2 .DNA without helicase bound interacts transiently with the nanoporeproducing short-lived blocks in current (<<1 second). DNA with helicasebound and active (ie. moving along the DNA strand under ATPase action)produces long characteristic blocks levels with stepwise changes incurrent as shown in FIG. 2 . Different DNA motifs in the nanopore giverise to unique current block levels.

For a given substrate, we observe a characteristic pattern of currenttransitions that reflects the DNA sequence (examples in FIGS. 3A-3B).

In the implementation shown in FIGS. 1A-1B, the DNA strand is sequencedfrom a random starting point as the DNA is captured with a helicase at arandom position along the strand. However, as long as the enzyme doesnot dissociate, the strands will all end in the same way at the 50Tleader (FIGS. 1A-1B). As FIG. 2 shows, we observe the samecharacteristic ending to most strands, with the current transitionsending in a long dwell time polyT level (FIGS. 3A-3B).

Salt Tolerance

Nanopore strand sequencing experiments of this type require ionic salts.The ionic salts are necessary to create a conductive solution forapplying a voltage offset to capture and translocate DNA, and to measurethe resulting sequence dependent current changes as the DNA passesthrough the nanopore. Since the measurement signal is dependent in theconcentration of the ions, it is advantageous to use high concentrationionic salts to increase the magnitude of the acquired signal. Fornanopore sequencing salt concentrations in excess of 100 mM KCl areideal, and salt concentrations of 1 M KCl and above are preferred.

However, many enzymes (including some helicases and DNA motor proteins)do not tolerate high salt conditions. Under high salt conditions theenzymes either unfold or lose structural integrity, or fail to functionproperly. The current literature for known and studied helicases showsthat almost all helicases fail to function above salt concentrations ofapproximately 100 mM KCl/NaCl, and there are no reported helicases thatshow correct activity in conditions of 400 mM KCl and above. Whilepotentially halophilic variants of similar enzymes from halotolerantspecies exist, they are extremely difficult to express and purify instandard expression systems (e.g. E. coli).

We surprisingly show in this Example that Hel308 from Mbu displays salttolerance up to very high levels of KCl. We find that the enzyme retainsfunctionality in salt concentrations of 400 mM KCl through to 2 M KCl,either in fluorescence experiments or in nanopore experiments (FIGS.4A-4D). FIGS. 4A-4C show the Hel308 Mbu DNA events at 400 mM KCl, 1 MKCl, and 2 M KCl salt conditions carried out using the same systemdescribed in FIGS. 1A-1B.

We observe similar movement across the range of salt concentrations. Asthe salt concentration is increased we observe an increase in thecurrent through the nanopore (I-open) at a fixed voltage. This reflectsthe increase in the conductivity of the solution and the increasednumber of ions flowing through the nanopore under the applied field. Inaddition we also observe an increase in the minimum to maximum range ofdiscrimination in the current levels of the DNA events (see FIGS. 4A-4Cenlargements and bottom right plot). We observe a ˜200% increase in DNAdiscrimination range as the salt concentration is increased from 400 mMKCl to 2M KCl (Table 6 below; FIG. 4D).

TABLE 6 Effect of increasing salt concentration on pore current and DNArange Salt (KCl) Open-pore current DNA (M) (pA) range (pA) 0.4 180 251.0 440 55 2.0 840 75Forward and Reverse Modes of Operation

Most helicases move along single-stranded polynucleotide substrates inuni-directional manner, moving a specific number of bases for eachNTPase turned over. Although FIGS. 1A-1B illustrate the use of thismovement to pull threaded DNA out of the nanopore, helicase movementcould be exploited in other manners to feed DNA through the nanopore ina controlled fashion. FIGS. 5A-5B illustrate the basic ‘forward’ and‘reverse’ modes of operation. In the forward mode, the DNA is fed intothe pore by the helicase in the same direction as the DNA would moveunder the force of the applied field. For Hel308 Mbu, which is a 3′-5′helicase, this requires capturing the 3′ end of the DNA in the nanoporeuntil a helicase contacts the top of the nanopore, and the DNA is thenfed into the nanopore under the control of the helicase with the fieldfrom the applied potential, finally exiting on the trans side of thebilayer. The reverse mode requires capturing the 5′ end of the DNA,after which the helicase proceeds to pull the threaded DNA back out ofthe nanopore against the field from the applied potential, finallyejecting it on this cis side of the bilayer. FIGS. 5A-5B show these twomodes of operation using Hel308 Mbu, and typical example DNA events.

Example 2

This Example illustrates the salt tolerance of a Hel308 helicase (Hel308MBu) using a fluorescence assay for testing enzyme activity.

A custom fluorescent substrate was used to assay the ability of thehelicase to displace hybridised dsDNA (FIG. 6A). As shown in 1) of FIG.6A, the fluorescent substrate strand (100 nM final) has a 3′ ssDNAoverhang, and a 40 base section of hybridised dsDNA. The major upperstrand has a carboxyfluorescein base at the 5′ end, and the hybrisedcomplement has a black-hole quencher (BHQ-1) base at the 3′ end. Whenhybrised the fluorescence from the fluorescein is quenched by the localBHQ-1, and the substrate is essentially non-fluorescent. 1 μM of acapture strand that is complementary to the shorter strand of thefluorescent substrate is included in the assay. As shown in 2), in thepresence of ATP (1 mM) and MgCl₂ (5 mM), helicase (100 nM) added to thesubstrate binds to the 3′ tail of the fluorescent substrate, moves alongthe major strand, and displaces the complementary strand as shown. Asshown in 3), once the complementary strand with BHQ-1 is fully displacedthe fluorescein on the major strand fluoresces. As shown in 4), anexcess of capture strand preferentially anneals to the complementary DNAto prevent re-annealing of initial substrate and loss of fluorescence.

Substrate DNA: 5′FAM-SEQ ID NO: 61 and SEQ ID NO: 62-BHQ1-3′.FAM=carboxyfluorescein and BHQ1=Black Hole Quencher-1

Capture DNA: SEQ ID NO: 62.

The graph in FIG. 6B shows the initial rate of activity in buffersolutions (10 mM Hepes pH 8.0, 1 mM ATP, 5 mM MgCl₂, 100 nM fluorescentsubstrate DNA, 1 μM capture DNA) containing different concentrations ofKCl from 400 mM to 2 M. The helicase works at 2 M.

Example 3

In this Example, three different Hel308 helicases were used, namelyHel308 Mhu (SEQ ID NO: 52), Hel308 Mok (SEQ ID NO: 29) and Hel308 Mma(SEQ ID NO: 45). All experiments were carried out as previouslydescribed in Example 1 under the same experimental conditions (pore=MspAB2, DNA=400mer SEQ ID NO: 59 and 60, buffer=400 mM KCl, 10 mM Hepes pH8.0, 1 mM dtt, 1 mM ATP, 0.1 mM MgCl₂). The results are shown in FIGS.7A-7C.

Example 4

This Example measures the internal binding capabilities of a number ofHel308 helicases using a fluorescence assay.

Custom fluorescent substrates were used to assay the ability of thehelicases to initiate on DNA lacking native 3′ ends, allowing them tosubsequently displace hybridised dsDNA (FIG. 8 ). As shown in Panel A ofFIG. 8 , the fluorescent substrate strand (50 nM final) has a 3′ ssDNAoverhang, and a 40 base section of hybridised dsDNA. The major upperstrands are modified with four consecutive “spacer 9” groups, either atthe 3′ end, or internally, at the junction between the overhang and thedsDNA (as a negative control). Furthermore, the major upper strand has acarboxyfluorescein base at the 5′ end, and the hybridised complement hasa black-hole quencher (BHQ-1) base at the 3′ end. When hybridised, thefluorescence from the fluorescein is quenched by the local BHQ-1, andthe substrate is essentially non-fluorescent. A capture strand (1 μM),that is complementary to the shorter strand of the fluorescentsubstrate, is included in the assay. In the presence of ATP (1 mM) andMgCl₂ (1 mM), a Hel308 helicase homologue (20 nM), added to thesubstrate containing 3′-terminal “spacer 9” groups, can bind to thessDNA overhang of the fluorescent substrate, move along the majorstrand, and displace the complementary strand as shown in Panel B. Oncethe complementary strand with BHQ-1 is fully displaced (Panel C) thefluorescein on the major strand fluoresces. An excess of capture strandpreferentially anneals to the complementary DNA to prevent re-annealingof initial substrate and loss of fluorescence (Panel D).

Substrate DNA: SEQ ID NO: 63 with a 5′ FAM; SEQ ID NO: 63 with a 5′ FAMand 3′ spacer ((spacer 9)₄); SEQ ID NOs: 64 (with a 5′ FAM) and 65separated by a spacer ((spacer 9)₄); and SEQ ID NO: 62 with a 3′ BHQ1.

Capture DNA: SEQ ID NO: 66.

A number of different Hel308 helicase homologues were investigated fortheir mid-binding abilities, these included Hel308 Mbu, Hel308 Csy,Hel308 Tga, Hel308 Mma, Hel308 Mhu, Hel308 Min, Hel308 Mig, Hel308 Mmaz,Hel308 Mac, Hel308 Mok, Hel308 Mth, Hel308 Mba and Hel308 Mzh. The graphin FIG. 9 shows the relative rates of Hel308-mediated dsDNA turnover,comparing 3′-unmodified DNA and 3′-“spacer 9” DNA in 400 mM NaCl, 10 mMHepes, pH 8.0, 1 mM ATP, 1 mM MgCl₂, 50 nM fluorescent substrate DNA, 1μM capture DNA. Several Hel308 homologues were observed to have greaterthan 20% relative rates of Hel308-mediated dsDNA turnover including,Hel308 Csy, Hel308 Tga, Hel308 Mma, Hel308 Mhu and Hel308 Min.

Example 5

This Example compares the use of two Hel308 helicases, Hel308 MBu andHel 308 Tga, and their ability to control the movement of intact longDNA strands (900 mer) through a nanopore. The general method andsubstrate employed throughout this Example are shown in FIG. 10 anddescribed in the description of the Figure above.

Materials and Methods

The DNA was formed by ligating a 50-polyT 5′ leader to a ˜900basefragment of PhiX dsDNA. The leader also contains a complementary sectionto which SEQ ID NO: 69 with a Chol-tag was hybridized to allow the DNAto be tethered to the bilayer. Finally the 3′ end of the PhiX dsDNA wasdigested with AatII digestion enzyme to yield a 4nt 3′-overhang of ACGT.

Sequencesused: SEQ ID NO: 67-900mer sense strand including 5′ leader andtether; SEQ ID NO: 68— anti-sense minus 4 base-pair leader 5′; and SEQID NO: 69 with several spacers and a Chol-tag at the 3′ end.

Buffered solution: 400 mM-2 NaCl, 10 mM potassium ferrocyanide, 10 mMpotassium ferricyanide, 100 mM Hepes, pH 8.0, 1 mM ATP, 1 mM MgCl₂,

Nanopore: MS-(B1-G75S-G77S-L88N-Q126R)8 (ONT Ref B2C)

Enzyme: Hel308 Mbu 1000 nM or Hel308 Tga 400 nM final.

Electrical experiments were set up as described in Example 1 in order toachieve a single pore inserted into a lipid bilayer. After achieving asingle pore in the bilayer, ATP (1 mM) and MgCl₂ (1 mM) were added tothe chamber. A control recording at +140 mV was run for 2 minutes. DNApolynucleotide SEQ ID NOs: 67, 68 and 69 (DNA=0.15 nM) were then addedand DNA events observed. Finally, Hel308 helicase (Mbu 1000 nM or Tga,400 nM) was added to the cis compartment of the electrophysiologychamber to initiate capture of the helicase-DNA complexes in the MspAnanopore. Experiments were carried out at a constant potential of +140mV.

Results and Discussion

The addition of Helicase-DNA substrate to MspA nanopores as shown inFIG. 10 produces characteristic current blocks as the helicase controlsthe translocation of the DNA through the pore. FIG. 11 shows exampleevent traces which indicate how the position of the 900 mer varied asthe Hel308 helicase homologue Mbu controlled the translocation of theDNA strand through the MspA pore. This helicase was found to mediatecontrol of DNA translocation, however, when the helicase detached fromthe DNA, the strand was observed to move back through the pore, owing tothe force exerted by the externally applied potential. In the case ofthe Hel308 helicase homologue Mbu, the 900mer strand slipped back alarge number of positions (approximately 100-200 bases) each time ahelicase disengaged. These rapid changes in position are indicated inFIG. 11 by dotted circles. For this experiment, where Hel308 helicasehomologue Mbu was used as the molecular motor, 32% of all of the eventsdetected were found to have read the entire length of the 900 mer strandsequence. FIG. 12 shows similar example event traces indicating how theposition of the 900 mer varied as the Hel308 helicase homologue Tgacontrolled the translocation of the DNA strand through the MspA pore.This enzyme exhibited an greater tendency to bind internally, than theMbu homologue, because when the Tga helicase disengages (indicated by achange in colour black to grey in FIG. 12 ), the DNA strand moves backthrough the pore by a relatively small distance (<50 bases). For thisexperiment, where Hel308 helicase homologue Tga was used as themolecular motor, 74% of all of the events detected were found to haveread the entire length of the 900 mer strand sequence. This means thatthe Tga helicase homologue can provide increased read lengths ofsingle-stranded DNA in comparison to the Mbu helicase homologue owing toits increased tendency to bind internally.

Example 6

This Example illustrates that by employing the Hel308 helicase homologueTga it is possible to control the translocation of a 5 kb strand of DNA.

A similar experimental procedure was followed to that described inExample 5. It was observed that by employing the Hel308 Tga it waspossible to detect the controlled translocation of an entire 5 kb strandof DNA through MS-(B1-G75S-G77S-L88N-Q126R)8. In an identical experimentusing Hel308 Mbu, it was not possible to detect translocation of anentire 5 kB strand.

Example 7

This example compares the enzyme processivity of Hel308 Mbu helicase(SEQ ID NO: 10) with Hel308 Mok (SEQ ID NO: 29) using a fluorescencebased assay.

A custom fluorescent substrate was used to assay the ability of thehelicase to displace hybridised dsDNA (FIG. 13 ). The fluorescentsubstrate (50 nM final) has a 3′ ssDNA overhang, and 80 and 33 base-pairsections of hybridised dsDNA (FIG. 13 Panel A, SEQ ID NO: 70). The majorlower “template” strand is hybridised to an 80 nt “blocker” strand (SEQID NO: 71), adjacent to its 3′ overhang, and a 33 nt fluorescent probe,labelled at its 5′ and 3′ ends with carboxyfluorescein (FAM) andblack-hole quencher (BHQ-1) bases, respectively (SEQ ID NO: 72). Whenhybridised, the FAM is distant from the BHQ-1 and the substrate isessentially fluorescent. In the presence of ATP (1 mM) and MgCl₂ (10mM), the helicase (20 nM) binds to the substrate's 3′ overhang (SEQ IDNO: 70), moves along the lower strand, and begins to displace the 80 ntblocker strand (SEQ ID NO: 71), as shown in FIG. 13 Panel B. Ifprocessive, the helicase displaces the fluorescent probe (SEQ ID NO: 72,labeled with a carboxyfluorescein (FAM) at its 5′ end a black-holequencher (BHQ-1) at its 3′ end) too (FIG. 13 Panel C). The fluorescentprobe is designed in such a way that its 5′ and 3′ ends areself-complementary and thus form a kinetically-stable hairpin oncedisplaced, preventing the probe from re-annealing to the template strand(FIG. 13 Panel D). Upon formation of the hairpin product, the FAM isbrought into the vicinity of the BHQ-1 and its fluorescence is quenched.A processive enzyme, capable of displacing the 80 mer “blocker” (SEQ IDNO: 71) and fluorescent (SEQ ID NO: 72, labeled with acarboxyfluorescein (FAM) at its 5′ end a black-hole quencher (BHQ-1) atits 3′ end) strands will therefore lead to a decrease in fluorescenceover time. However, if the enzyme has a processivity of less than 80 ntit would be unable to displace the fluorescent strand (SEQ ID NO: 72,labeled with a carboxyfluorescein (FAM) at its 5′ end a black-holequencher (BHQ-1) at its 3′ end) and, therefore, the “blocker” strand(SEQ ID NO: 71) would reanneal to the major bottom strand (FIG. 13 PanelE, SEQ ID NO: 70).

Additional custom fluorescent substrates were also used for controlpurposes. The substrate used as a negative control was identical to thatof the one described in FIG. 13 but lacking the 3′ overhang (FIG. 14Panel A, (SEQ ID NOs: 71, 72 (labeled with a carboxyfluorescein (FAM) atits 5′ end a black-hole quencher (BHQ-1) at its 3′ end) and 73)). Asimilar substrate to that described in FIG. 13 but lacking the 80 basepair section, used as a positive control for active, but not necessarilyprocessive, helicases (FIG. 14 Panel B, (SEQ ID NOs: 72 (labeled with acarboxyfluorescein (FAM) at its 5′ end a black-hole quencher (BHQ-1) atits 3′ end) and 74)).

FIG. 15 shows a graph of the time-dependent fluorescence changes upontesting Hel308 Mbu helicase (SEQ ID NO: 10) and Hel 308 Mok helicase(SEQ ID NO: 29) against the processivity substrate shown in FIG. 13 inbuffered solution (400 mM NaCl, 10 mM Hepes pH 8.0, 1 mM ATP, 10 mMMgCl₂, 50 nM fluorescent substrate DNA (SEQ ID NOs: 70, 71 and 72(labeled with a carboxyfluorescein (FAM) at its 5′ end a black-holequencher (BHQ-1) at its 3′ end). The decrease in fluorescence exhibitedby Hel308 Mok denotes the increased processivity of these complexes ascompared to Hel308 Mbu (SEQ ID NO: 10). FIG. 16 shows positive controlsdemonstrating that all helicases were indeed active, as denoted by afluorescence decrease for all samples.

SEQ ID NOs: 10, 13, 16, 19, 22, 25, 28, 29, 32, 33, 34, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54 and 55 (Table 4) are aligned below*                   1                                                                                            95 10 Hel308 Mbu   (1) --------------------------------MMIRELDIPRDIIGFYEDSGIKELYPPQAEAIEMGLLE-KKNLLAAIPTASGKTLLAELAMIK53 Hel308 Afu   (1) ----------------------------- MKVEELAESISSYAVGILKEEGIEELFPPQAEAVEKVFS--GKNLLLAMPTAAGKTLLAEMAMVR22 Hel308 Csy   (1) --------------------------------MRISELDIPRPAIEFLEGEGYKKLYPPQAAAAKAGLTD-GKSVLVSAPTASGKTLIAAIAMIS75 Hel308 Dth   (1) MPGVDELLQQMGQGDLQGLSTVAVKEIPAREAEFSGIEGLPPPLKQALTESGIENFYTHQARAVNLVRK--GRSVVTATPTASGKSLIYNIPVLE48 Hel308 Fac   (1) ----------------------------------MKLSEITPSEFLKVTDNNDFTLYEHQEEAVAKLREN--KNVIVSVPTASGKTLIGYISIYD 19 Hel308 Hla   (1) -------------------------------MQPSSLSGLPAGVGEALEAEGVAELYPPQEAAVEAGVAD-GESLVAAVPTASGKTLIAELAMLS 55 Hel308 Hpa   (1) -------------------------------MNVADLTGLPDGVPEHFHAQGIEELYPPQAEAVEAGITE-GESVVASIPTASGKTFIAELAMLS 54 Hel308 Htu   (1) -------------------------------MNLEELTGLPPGATDHFRGEGIEELYPPQADAVEAGATD-GENLVAAVPTASGKTMIAALSMLS 16 Hel308 Hvo   (1) -------------------------------MRTADLTGLPTGIPEALRDEGIEELYPPQAEAVEAGLTD-GESLVAAVPTASGKTLIAELAMLS 39 Hel308 Mac   (1) --------------------------------MKIESLDLPDEVKRFYENSGIPELYPPQAEAVEKGLLE-GKNLLAAIPTASGKTLLAELAMLK 38 Hel308 Mba   (1) --------------------------------MKIESLDLPDEVKQFYLNSGIMELYPPQAEAVEKGLLE-GRNLLAAIPTASGKTLLAELAMLK 47 Hel308 Mbo   (1) --------------------------------MQIQDLAIPEPLRQQYLGLGIRELYPPQAACVERGLLD-GKNLLVAIPTASGKTLIAEMAMHR 44 Hel308 Mev   (1) --------------------------------METGKLELPEYVIQFYLDTGIEKLYPPQAEAVEKGLLD-NKNLLAAIPTASGKTLISELAMLK 49 Hel308 Mfe   (1) --------------------------------------MPTNKILEILKDFGIEELRPPQKKALEKGLLDKNKNFLISIPTASGKTLIGEMALIN 28 Hel308 Mfr   (1) ------------------------------------DLSLPKAFIQYYKDKGIESLYPPQSECIENGLLD-GADLLVAIPTASGKTLIAEMAMHA 52 Hel308 Mhu   (1) --------------------------------MEIASLPLPDSFIRACHAKGIRSLYPPQAECIEKGLLE-GKNLLISIPTASGKTLLAEMAMWS 32 Hel308 Mig   (1) -------------------------------------MQKYSHVFEVLKENGIKELRPPQKKVIEKGLLNKEKNFLICIPTASGKTLIGEMALIN 51 Hel308 Min   (1) -------------------------------------------MDEILKFLGIKELRPPQKKALELGILDKKKNFLISIPTGAGKTVIAEMALIN 45 Hel308 Mma   (1) -----------------------------------------MHVLDLLKENKITELRPPQKKVIDEGLFDKTKNFLICIPTASGKTLIGEMALLN 40 Hel308 Mmah  (1) --------------------------------MKIEELDLPSEAIEVYLQAGIEELYPPQADAVEKGLLQ-GENLLAAIPTASGKTLLAEMAMLK 76 Hel308 Mmar  (1) -------------------------------MDVADLPGVPEWLPDHLRDDGIEELYPPQAEAVEAGVTE-GENLVASIPTASGKTLIAELAMLS 41 Hel308 Mmaz  (1) --------------------------------MKIESLDLPDEIKRFYENSGILELYPPQAEAVEKGLLE-GKNLLAAIPTASGKTLLAELAMLK 29 Hel308 Mok   (1) ----------------------------------------MLMLMEVLKENGIAELRPPQKKVVEGGLLNKNKNFLICIPTASGKTLIGEMAFIN 42 Hel308 Mth   (1) -----------------------------MLTIRDLIRWLPESVIELYEALGIDELYPPQAEAIERGLLD-GRNMIISVPTAAGKTLLAELAMLR 43 Hel308 Mzh   (1) --------------------------------MNINNLNLPEKVKKYYTDTGIVDLYPPQREAVDKGLLD-GENIVAAIPTASGKTLLAELCMLK 46 Hel308 Nma   (1) -------------------------------MNVEELSGLPPGARSHFQEQGIEELYPPQAEAVEAGATE-GENLVAAVPTASGKTMIAALSMLS 77 Hel308 Nth   (1) --------------------------------MSETFYLLSERMQKKIWEMGWDEFTPVQDKTIPIVMNT-NKDVVVSSGTASGKTEAVFLPILS 13 Hel308 Pfu   (1) -----------------------------MRVDELR---VDERIKSTLKERGIESFYPPQAEALKSGILE-GKNALISIPTASGKTLIAEIAMVH 25 Hel308 Sso   (1) -------------------------MSLELEWMPIEDLKLPSNVIEIIKKRGIKKLNPPQTEAVKKGLLE-GNRLLLTSPTGSGKTLIAEMGIIS 34 Hel308 Tba   (1) ---------MLSTKPKAYKRFSPIG--YAMQVDELSKFGVDERIIRKIKERGISEFYPPQAEALRSGVLN-GENLLLAIPTASGKTLVAEIVMLH 33 Hel308 Tga   (1) -----------------------------MKVDELP---VDERLKAVLKERGIEELYPPQAEALKSGALE-GRNLVLAIPTASGKTLVSEIVMVN 37 Hel308 Tsi   (1) ---------MKLNKLKSYINAFLLGMVMSMKVDELKSLGVDERILRLLRERGIEELYPPQADALKTEVLK-GKNLVLAIPTASGKTLVAEIVMIN 50 Hel308 Mja   (1) ----------------------------------------MDKILEILKDFGIVELRPPQKKALERGLLDKNKNFLISIPTASGKTLIGEMALIN 78 Consensus    (1)                                        LP  V   L E GI ELYPPQAEAVE GLLD GKNLLIAIPTASGKTLIAELAML                     96                                                                                          190    Hel308 Mbu  (63) AIREGG------KALYIVPLRALASEKFERFK-ELAP----FGIKVGISTGDLDSRADWLGVNDIIVATSEKTDSLLRNGTSWMD-------EIT    Hel308 Afu  (64) EAIKGG------KSLYVVPLRALAGEKYESFK-KWEK----IGLRIGISTGDYESRDEHLGDCDIIVTTSEKADSLIRNRASWIK-------AVS    Hel308 Csy  (63) HLSRNR-----GKAVYLSPLRALAAEKFAEFGKIGGIPL-GRPVRVGVSTGDFEKAGRSLGNNDILVLTNERMDSLIRRRPDWMD-------EVG    Hel308 Dth  (94) SIINDP----ASRALYLFPLKALTRDQLTSLEEFARLLAGKVHVDSAVYDGDTDPQARARIRSKPPNILLTNPDMLHRSFLPYHRSWQKFFSALK    Hel308 Fac  (60) TYLKGK------KSMYIVPLRSLAMEKFSELL-SLRN----LGVKVTMSIGDYDVPPSFVKNYDVIIATSERADSMLHRDPDILN-------YFG    Hel308 Hla  (64) SIERGG------KALYIVPLRALASEKKTEFE-RWEE----FGVTVGVSTGNYESDGEWLATRDIIVATSEKVDSLIRNGAPWID-------DLT    Hel308 Hpa  (64) SVARGG------KALYIVPLRALASEKKEEFE-EFEQ----YGVSIGVSTGNYESDGDWLASRDIIVATSEKVDSLVRNGAKWID-------DLS    Hel308 Htu  (64) AVQRGG------KALYIVPLRALASEKKEEFE-AYEE----FGVTTGVTTGNYESTDDWLATKDIIVATSEKVDSLVRNGADWLS-------ELT    Hel308 Hvo  (64) SVARGG------KALYIVPLRALASEKKAEFE-RWEE----YGIDVGVSTGNYESDGEWLSSRDIIVATSEKVDSLVRNNAAWMD-------QLT    Hel308 Mac  (63) SVLAGG------KALYIVPLRALASEKFRRFQ-DFSE----LGIRVGISTGDYDRRDEGLGINDIIVATSEKTDSLLRNETAWMQ-------EIS    Hel308 Mba  (63) SILAGG------KALYIVPLRALASEKFRRFR-EFSE----LGIRVGISTGDYDLRDEGLGVNDIIVATSEKTDSLLRNETVWMQ-------EIS    Hel308 Mbo  (63) HIANGG------KCLYIVPLKALASEKYEEFG-NK-------GVKVGLSTGDLDRRDDALGKNDIIVATSEKVDSLLRNGARWIP-------DIT    Hel308 Mev  (63) SISNGG------KCLYIVPLRALASEKFERFK-QFSS----IGVNIGISTGDFDSTDEWLGSNDIIVATSEKADSLLRNETSWMK-------DIT    Hel308 Mfe  (58) HLLDENKNPTNKKGIFIVPLKALASEKYEEFKNKYER----YGLRVALSIGDYD-EDEDLSRYHLIITTAEKLDSLWRHKIDWID-------DVS    Hel308 Mfr  (59) AIARGG------MCLYIVPLKALATEKAQEFK-GK-------GAEIGVATGDYDQKEKRLGSNDIVIATSEKVDSLLRNGVPWLS-------QVT    Hel308 Mhu  (63) RIAAGG------KCLYIVPLRALASEKYDEFS-KKG------VIRVGIATGDLDRTDAYLGENDIIVATSEKTDSLLRNRTPWLS-------QIT    Hel308 Mig  (59) HLLDENKTPTNKKGLFIVPLKALASEKYEEFKRKYEK----YGLKVALSIGDYD-EKEDLSSYNIIITTAEKLDSLMRHEIDWLN-------YVS    Hel308 Min  (53) HLLLDK----GKKGVYIVPLKALASEKYEEFKKKYEK----FGVRVALSIGDYD-EDEDLENYDLIITTAEKFDSLWRHGIKLS--------DIS    Hel308 Mma  (55) HILDENKNLTGKKGLFIVPLKALANEKFDEFREKYEK----YGIKVGLSIGDFD-TKENLSKFHIIITTSEKLDSLMRHNVEWIN-------DVS   Hel308 Mmah  (63) AIKKGG------KALYIVPLRALASEKFRDFK-RFES----LGIKTAISTGDFDSRDEWLGSNDIIVATSEKTDSLLRNSTPWMK-------DIT   Hel308 Mmar  (64) SVARGG------KALYIVPLRALASEKQADFE-EFEQ----YGLDIGVSTGNYESEGGWLADKDIVVATSEKVDSLVRNDAPWIE-------DLT   Hel308 Mmaz  (63) SVLNGG------KALYIVPLRALASEKFRRFQ-EFSV----LGMRVGISTGDYDRRDEGLGINDIIVATSEKTDSLLRNETAWMQ-------EIS    Hel308 Mok  (56) HLLDNNKTPTNKKGLFIVPLKALANEKYEEFKGKYEK----YGLKIALSIGDFD-EKEDLKGYDLIITTAEKLDSLIRHKVEWIK-------DIS    Hel308 Mth  (66) GALSGK------RSLYIVPLRALASEKFESFS-RFSK----LGLRVGISTGDFEKRDERLGRNDIIIATSEKADSLIRNGASWVR-------RIG    Hel308 Mzh  (63) SIGMGG------KCLYIVPLKALASEKYSRFR-EFES----LGIKVGIATGDLDSREEWLGKNDIIIATSEKVDSLLRNESSWMK-------EIN    Hel308 Nma  (64) AVQRGG------KALYIVPLRALASEKKAEFD-AYEE----FGVTTGVATGNYESTSEWLATKDIIVATSEKVDSLVRNGADWLS-------DLT    Hel308 Nth  (63) QIEKDAT--KDLKILYISPLKALINDQFERIIKLCEKSY-IPIHRWHGDVNQNKKKQLTKNPAGILQITPESIESLFINRTNELNYIL---SDIE    Hel308 Pfu  (63) RILTQG-----GKAVYIVPLKALAEEKFQEFQ-DWEK----IGLRVAMATGDYDSKDEWLGKYDIIIATAEKFDSLLRHGSSWIK-------DVK    Hel308 Sso  (70) FLLKNG-----GKAIYVTPLRALTNEKYLTFK-DWEL----IGFKVAMTSGDYDTDDAWLKNYDIIITTYEKLDSLWRHRPEWLN-------EVN    Hel308 Tba  (84) KLFTGG-----GKAVYLVPLKALAEEKYREFK-TWED----LGVRVAVTTGDYDSSEEWLGKYDIIIATSEKFDSLLRHKSRWIR-------DVT    Hel308 Tga  (63) KLIQEG-----GKAVYLVPLKALAEEKYREFK-EWEK----LGLKVAATTGDYDSTDDWLGRYDIIVATAEKFDSLLRHGARWIN-------DVK    Hel308 Tsi  (86) KILREG-----GKTVYLVPLKALAEEKYKEFK-FWEK----LGIRIAMTTGDYDSTEEWLGKYDIIIATSEKFDSLLRHKSPWIK-------DIN    Hel308 Mja  (56) HLLDGNKNPTNKKGIFIVPLKALASEKYEEFKSKYER----YGLRIALSIGDYD-EDEDLSKYHLIITTAEKLDSLWRHKIDWIN-------DVS    Consensus   (96)  IL GG      KALYIVPLRALASEKY EFK  FE      GVRVGISTGDYD  DEWLG  DIIVATSEKVDSLLRN   WI        DIT                     191                                                                                         285    Hel308 Mbu (140) TVVVDEIHLLDSKNRGPTLEVTITKLMRLNPD----VQVVALSATVGNAREMADWLG---AALVLSEWRPTDLHEGVLFGDAINFPG-SQKKIDR    Hel308 Afu (141) CLVVDEIHLLDSEKRGATLEILVTKMRRMNKA----LRVIGLSATAPNVTEIAEWLD---ADYYVSDWRPVPLVEGVLCEGTLELFD----GAFS    Hel308 Csy (145) LVIADEIHLIGDRSRGPTLEMVLTKLRGLRSS----PQVVALSATISNADEIAGWLD---CTLVHSTWRPVPLSEGVYQDGEVAMGDGSRHEVAA    Hel308 Dth (185) YIVVDEVHTYRG-VMGSNMAWVFRRLRRICAQYGREPVFIFSSATIANPGQLCSALTGHEPEVIQKGGAPAGKKHFLLLDPEMQGAAQS------    Hel308 Fac (137) LVIIDEIHMISDPSRGPRLETVISSLLYLNPE----ILLLGLSATVSNIQEIAEWMN---AETVVSNFRAVPLETGIIFKGNLITDG--------   Hel308 Hla (141) CVVSDEVHLVDDPNRGPTLEVTLAKLRKVNPG----LQTVALSATVGNADVIAEWLD---AELVESDWRPIDLRMGVHFGNAIDFADGSKREVPV    Hel308 Hpa (141) CVVADEVHLVNDAHRGPTLEVTLAKLRRVNPD----LQTVALSATVGNAGEMADWLD---ATLVDSTWRPIDLRKGVLYGQALHFDDGTQQELAR    Hel308 Htu (141) CVVSDEVHLIDDRNRGPTLEVTLAKLRRLNPG----MQVVALSATVGNADEIADWLD---ASLVDTDWRPIDLQMGVHYGNALNFDDGSTREVPV    Hel308 Hvo (141) CVVADEVHLVDDRHRGPTLEVTLAKLRRLNTN----LQVVALSATVGNAGVVSDWLD---AELVKSDWRPIDLKMGVHYGNAVSFADGSQREVPV    Hel308 Mac (140) VVVVDEVHLIDSADRGPTLEVTLAKLRKMNPF----CQILALSATVGNADELAAWLD---AELVLSEWRPTDLMEGVFFDGTFFCKD-KEKLIEQ    Hel308 Mba (140) VVVADEVHLIDSPDRGPTLEVTLAKLRKMNPS----CQILALSATVGNADELAVWLE---AELVVSEWRPTELLEGVFFNGTFYCKD-REKTVEQ    Hel308 Mbo (137) LVVIDEIHLIDSPDRGPTLEMVIAKMRSKNPG----MQLIGLSATIGNPKVLAGWLD---AELVTSSWRPVDLRQGVFYDNRIQFAE-RMRPVKQ    Hel308 Mev (140) TIVVDEIHLLDSADRGPTLEITIAKLLRLNPN----SQIIGLSATIGNAEEIAGWLD---AELVQSQWRPIELYEGVFLEDNINFKQ-SQKPIKN    Hel308 Mfe (141) VVVVDEIHLINDESRGGTLEILLTKLKKFN------IQIIGLSATIGNPEELANWLN---AELIVDDWRPVELKKGIYKNGIIEFINGE-----N    Hel308 Mfr (133) CLVVDEVHLIDDESRGPTLEMVITKLRHASPD----MQVIGLSATIGNPKELAGWLG---ADLITSDWRPVDLREGICYHNTIYFDN-EDKEIPA    Hel308 Mhu (138) CIVLDEVHLIGSENRGATLEMVITKLRYTNPV----MQIIGLSATIGNPAQLAEWLD---ATLITSTWRPVDLRQGVYYNGKIRFSD-SERPIQG    Hel308 Mig (142) VAIVDEIHMINDEKRGGTLEVLLTKLKNLD------VQIIGLSATIGNPEELAEWLN---AELIIDNWRPVKLRKGIFFQNKIMYLNGA-----C    Hel308 Min (131) VVVVDEIHVIGDSERGGTLEVLLTKLKELD------VQIIGLSATIGNPEELSEWLN---AELLLDNWRPVELRKGIYREGVIEYLDGE------   Hel308 Mma (138) LAVIDEIHLIGDNERGGTLEVILTKLKNLN------AQIVGLSATIGNPEELSNWLN---AKLIVDGWRPVELKKGIYFENELEFLKNP-----A   Hel308 Mmah (140) AVIVDEVHLLDSANRGPTLEVTLAKLKRLNPG----AQVVALSATVGNAMEIAQWLE---AKLVLSEWRPTYLHEGIFYGDAINFDE-DQTFIER   Hel308 Mmar (141) CVVTDEVHLVDDGERGPTLEVTLAKLRRLNPD----LQTVALSATIGNAEALATWLD---AGLVDSDWRPIDLQKGVHYGQALHLEDGSQQRLSV   Hel308 Mmaz (140) VVVADEVHLIDSPDRGPTLEITLSKLRRMNPS----CQVLALSATVGNADELAAWLD---AELVLSEWRPTDLMEGVFYNGIFYCKD-KEKPVGQ    Hel308 Mok (139) VVVIDEIHLIGDESRGGTLEVLLTKLKTKKT-----IQIIGLSATIGNPEELAKWLN---AELIVDEWRPVKLKKGIGYGNKIMFIDDNGNTINE    Hel308 Mth (143) VLVVDEIHLLDSANRGPTLEMTMTKLMHLNPE----MQVIGLSATIANGREIADWIK---GEIVSSDWRPVRLREGVLLEDRLVFPD-GEIQLEN    Hel308 Mzh (140) TVVADEVHLLNSVNRGPTLEITLAKLIHLNPG----SQIIALSATIGNPEDIAGWLG---ARLVVSEWRPTDLYEGILLDGLLHIGN-IKKDIQD    Hel308 Nma (141) CVVSDEVHLIDDRNRGPTLEVTLAKLRRLNPQ----LQVVALSATVGNADELADWLD---AELVDTDWRPIDLQMGVHYGNALNFDDGETREVPV    Hel308 Nth (152) FIIIDELHAFLDNERGVHLRSLLSRLENYIKEK---PRYFALSATLNNFKLIKEWIN---YNDIKNVEIIDSNEDDKDLLLSLMHFDKGKDYKKP    Hel308 Pfu (141) ILVADEIHLIGSRDRGATLEVILAHMLGKA-------QIIGLSATIGNPEELAEWLN---AELIVSDWRPVKLRRGVFYQGFVTWEDGSIDRFSS    Hel308 Sso (148) YFVLDELHYLNDPERGPVVESVTIRAKRRN--------LLALSATISNYKQIAKWLG---AEPVATNWRPVPLIEGVIYPERKKKEYNVIFKDNT    Hel308 Tba (162) LIVADEIHLLGSYDRGATLEMILSHMLGKA-------QILGLSATVGNAEELAEWLN---AKLVVSDWRPVKLRKGVFAHGQLIWEDGKVDKFPP    Hel308 Tga (141) LVVADEVHLIGSYDRGATLEMILTHMLGRA-------QILALSATVGNAEELAEWLD---ASLVVSDWRPVQLRRGVFHLGTLIWEDGKVESYPE    Hel308 Tsi (164) LVIADEIHLLGSYDRGATLEMILAHLDDKA-------QILGLSATVGNAEEVAEWLN---ADLVMSEWRPVALRKGVFYHGELFWEDGSIERFPT    Hel308 Mja (139) VVVVDEIHLINDETRGGTLEILLTKLKEFN------VQIIGLSATIGNPDELAEWLN---AELIVDDWRPVELKKGIYKNEAIEFINGEIREIKA    Consensus  (191) VVVVDEIHLI D  RGPTLEVLLAKLR LNP     LQIIALSATIGNAEELAEWL    AELVVSDWRPVDLR GVFY   L F D     I                     286                                                                                         380    Hel308 Mbu (227) LEK-----DDAVNLVLDTIKAEGQ-----CLVFESSRRNCAGFAKTASS---KVAKILDNDIMIKLAGIAEEVES--TGETDTAIVLANCIRKGV    Hel308 Afu (225) TSRR----VKFEELVEECVAENGG-----VLVFESTRRGAEKTAVKLSA---ITAKYVEN------EGLEKAILE--ENEGEMSRKLAECVRKGA    Hel308 Csy (233) TGGG-----PAVDLAAESVAEGGQ-----SLIFADTRARSASLAAKASA---VIPEAKGADAAKLAAAAKKIISS--GGETKLAKTLAELVEKGA    Hel308 Dth (273) ----------AIRVLQKALELGLR-----TIVYTQSRKMTELIAMWASQRAGRLKKYISAYRAGFLPEQRREIEQKLASGELLAVVSTSALELGI    Hel308 Fac (217) -EKKHLGRDDEVSLIKESIESGGQ-----ALVFRNSRRNAEKYAQSMVN----------------FFDFQNDFEKLEIPPDLFNEAQANMVAHGV    Hel308 Hla (229) ERGE----DQTARLVADALDTEEDGQGGSSLVFVNSRRNAESSARKLTD---VTGPRLTDDERDQLRELADEIRS--GSDTDTASDLADAVEQGS    Hel308 Hpa (229) -GNE----KETAALVRDTLEDGGS-----SLVFVNSRRNAEAAAKRLAD---VTKTHLTDDERRDLLDIADQIRD--VSDTETSDDLATAIEKGA    Hel308 Htu (229) EGSE----KQEAALVRDILREGGS-----SLVFVNSRRNAEGAAKRLGQ---VSSREITEDERAELAELADDIRD--DSDTETSADLADCVERGA    Hel308 Hvo (229) GRGE----RQTPALVADALEGDGEGDQGSSLVFVNSRRNAESAARRMAD---VTERYVTGDERSDLAELAAEIRD--VSDTETSDDLANAVAKGA    Hel308 Mac (227) PTK-----DEAINLVLDTLREGGQ-----CLVFESSRKNCMGFAKKATS---AVKKTLSAEDKEKLAGIADEILE--NSETDTASVLASCVRAGT    Hel308 Mba (227) STK-----DEAVNLALDTLKKDGQ-----CLVFESSRKNCMAFAKKAAS---TVKKTLSAEDRNALAGIADEILE--NSETDTSTNLAVCIRSGT    Hel308 Mbo (224) VSKN----YDDLNLCLDTIAEGGQ-----CLVFVSSRRNAEAFAKRAAG---AIKSEDA-----ALAACAERLLE--GTPTEMVKTLAACVAKGA    Hel308 Mev (227) IVK-----DTAVNLVLDTIDENGQ-----CLVFESSRRNCAGFAKKAKS---KVGKSLDKGLLAELNNIAEEVLE--TSDTETTKELASCIKRGT    Hel308 Mfe (222) REIKAINNNDIYNLVVDCVKDGGC-----CIVFCNTKRGAVNEAKKLN-----LKKFLTNEEKRKLKEVAEEILSILEPPTEMCKTLAECILNGS    Hel308 Mfr (220) PAK-----TEDINLLLDCVADGGQ-----CLVFVSSRRNAEGYAKRAAT---ALKCSHA-----ALDSIAEKLEA--AAETDMGRVLATCVKKGA    Hel308 Mhu (225) KTK-----HDDLNLCLDTIEEGGQ-----CLVFVSSRRNAEGFAKKAAG---ALKAGSP-----DSKALAQELRR--LRDRDEGNVLADCVERGA    Hel308 Mig (223) KELPNFSNNPMLNLVLDCVKEGGC-----CLVFCNSKNGAVSEAKKLN-----LKKYLSNSEKYELQKLKEEILSILDPPTETCKTLAECLEKGV    Hel308 Min (211) -------VKECQDIVKEVVKDNGS-----VIIFCPTKKKAENRALSLD-----LSDLLKKSEKRKLEEISEELLSLFDPPTELCKKLASCVRKGI    Hel308 Mma (219) KKIKQVSRNNLTDLIVDSVEEKGS-----CLIFCNSKRNAVGEAKKHN-----LAKYLTRTEQHELNKLSEEILSILDRPVETCKALSKCIQNGV   Hel308 Mmah (227) RHK-----EDSVNLVIDTVIQGGQ-----CLVFDSSRRNCVGFAKKCAP---AVGELLDRQNRNELEEVAKEVLE--NGETKLTETLAYCIKKGV   Hel308 Mmar (229) QNNE----KQTAAIVRDTLEDDGS-----TLVFVNSRRNAEAAAGRLAN---TVRPHLSTEERDQLADIAEEIRD--VSDTETSDDLADAVADGA   Hel308 Mmaz (227) PTK-----DEAVNLVLDTIKEGGQ-----CLVFESSRKNCMGFAKKAVS---AVKKTLSNEDRETLAGIADEIIE--NSETDVSSVLATCVRSGT    Hel308 Mok (226) VIVDEISKNNMFNLVVDSILKDGS-----CIIFCNSKRGAVGEAKKLN-----LKKYLSPDEISELRHLKEEVLSVLDNPTKTCKDLAECIEKGV    Hel308 Mth (230) RNR-----DPVLNLVLDTVDQGGQ-----MLIFESTRRNAESMAKKVSG---ALQESGE------TIELAERLS----GEGKTAKKLAMCLRHGA    Hel308 Mzh (227) ESR-----DDAVNLVIDTVKDKGQ-----CLVFESSRRNCMGFAKKAGK---WVSKILDEHDTIQLKSLSQEIGE--AGETEIADVLSRCVRQGV    Hel308 Nma (229) EAGE----KQEAALVRDILQEGGS-----SLVFVNSRRNAEAAARRLGQ---VSSRELTAGEQNDLAALATEIRE--DSDTETSQDLADCVERGA    Hel308 Nth (241) ----------ID-LYQDLRELTKN---VHSLIFCNSRAEVEETTLYLNR---LANREVNTELYLAHHSSIDKKER-EYVEKTMANSKSPKSVVTT    Hel308 Pfu (226) ----------WEELVYDAIRKKKG-----ALIFVNMRRKAERVALELSK---KVKSLLTKPEIRALNELADSLE----ENPTNEKLAKAIRGGV    Hel308 Sso (232) TKKVHG-DDAIIAYTLDSLSKNGQ-----VLVFRNSRKMAESTALKIAN---YMNFVSLDEN--ALSEILKQLDDIEEGGSDEKELLKSLISKGV    Hel308 Tba (247) Q---------WDSLVIDAVKKGKQ-----ALVFVNTRRSAEKEAGMLGK---KVRRLLTKPEARRLKELAESLE-----SNPTNDKLKEVLVNGA    Hel308 Tga (226) N---------WYSLVVDAVKRGKG-----ALVFVNTRRSAEKEALALSK---LVSSHLTKPEKRALESLASQLE-----DNPTSEKLKRALRGGV    Hel308 Tsi (249) Q---------WDSLVIDALKKGKQ-----ALVFVNTRRSAEKEALLLAG---KIQRFLTKPEERKLKQLADGLD-----TTPTNQKLKEALTKGV    Hel308 Mja (225) VDN-----NDIYNLVVDCVKEGGC-----CLVFCNTKRNAVNEAKKLN-----LKKFLTEEEKIRLKEIAEEILSILEPPTEMCKTLAECILNGS    Consensus  (286)              LVLDTV EGGQ      LVF NSRRNAE  AKKLA     V K LT  E   L  LAEEI      ETETS  LA CV KG                     381                                                                                         475    Hel308 Mbu (307) AFHHAGLNSNH-----------RKLVENGFRQNLIKVISSTPTLAA-------------------------------------------------   Hel308 Afu (300) AFHHAGLLNGQ-----------RRVVEDAFRRGNIKVVVATPTLAA-------------------------------------------------   Hel308 Csy (313) AFHHAGLNQDC-----------RSVVEEEFRSGRIRLLASTPTLAA-------------------------------------------------   Hel308 Dth (353) DIGHLDLCLLVGYPGSVMATMQRGGRVGRSGRDSAIMLIGHEDALDQYLLRNPREFFSLEPESAVINPDNPSIMRRHLVCAAAEKPIALQEMMLD    Hel308 Fac (290) MFHHAGLSNDQ-----------RTMIEKLFKQGYIKILTATPTLAA-------------------------------------------------   Hel308 Hla (315) AFHHAGLRSED-----------RARVEDAFRDRLIKCISATPTLAA-------------------------------------------------   Hel308 Hpa (309) AFHHAGLASDH-----------RSLVEDAFRDKLIKVISATPTLAA-------------------------------------------------   Hel308 Htu (310) AFHHAGLSSTQ-----------RSLVEDAFRDRLLKVISATPTLAA-------------------------------------------------   Hel308 Hvo (315) AFHHAGLAAEH-----------RTLVEDAFRDRLIKCICATPTLAA-------------------------------------------------   Hel308 Mac (307) AFHHAGLTSPL-----------RELVETGFREGYVKLISSTPTLAA-------------------------------------------------   Hel308 Mba (307) AFHHAGLTTPL-----------RELVEDGFRAGRIKLISSTPTLAA-------------------------------------------------   Hel308 Mbo (300) AFHHAGLSRKE-----------RSIVEEAFRKNLLKCISSTPTLAA-------------------------------------------------   Hel308 Mev (307) AFHHAGLNSAQ-----------RKIVEDNFRNNKIKVISSTPTLAA-------------------------------------------------   Hel308 Mfe (307) AFHHAGLTYQH-----------RKIVEDAFRNKLIKVICCTPTLSV-------------------------------------------------   Hel308 Mfr (295) AFHHAGMNRMQ-----------RTLVEGGFRDGFIKSISSTPTLAA-------------------------------------------------   Hel308 Mhu (300) AFHHAGLIRQE-----------RTIIEEGFRNGYIEVIAATPTLAA-------------------------------------------------   Hel308 Mig (308) AFHHAGLTYEH-----------RKIVEEGFRNKLIKVICCTPTLSA-------------------------------------------------   Hel308 Min (289) AFHHSGLTYEH-----------RKIIEKAFRERILKVICSTTTLAF-------------------------------------------------   Hel308 Mma (304) AFHHAGLTYKH-----------RKIVEDGFRNRLIKVICCTPTLSA-------------------------------------------------  Hel308 Mmah (307) AFHHAGLNSAH-----------RRIVEDAFRNNLIKMICSTPTLAA-------------------------------------------------  Hel308 Mmar (310) AFHHAGLSRGH-----------RELVEDAFRDRLVKVVCATPTLAA-------------------------------------------------  Hel308 Mmaz (307) AFHHAGLTTPL-----------RELVENGFREGRIKIISSTPTLAA-------------------------------------------------   Hel308 Mok (311) AFHHAGLTYEQ-----------RKIVEEGFRKKLIKAICCTPTLSA-------------------------------------------------   Hel308 Mth (302) AFHHAGLLPEQ-----------RRLIELGFRQNVVKVIACTPTLAA-------------------------------------------------   Hel308 Mzh (307) AFHHAGLNSEH-----------RRMVEEGFRKNLIKMISSTPTLAA-------------------------------------------------   Hel308 Nma (310) AFHHAGLSSTQ-----------RSLVEDAFRDRLLKVISATPTLAA-------------------------------------------------   Hel308 Nth (318) SSLELGIDIGA-----------IDYVVQIDDTHTVSSLKQRLGRSG-------------------------------------------------   Hel308 Pfu (298) AFHHAGLGRDE-----------RVLVEENFRKGIIKAVVATPTLSA-------------------------------------------------   Hel308 Sso (316) AYHHAGLSKAL-----------RDLIEEGFRQRKIKVIVATPTLAA-------------------------------------------------   Hel308 Tba (320) AFHHAGLGRAE-----------RTLIEDAFREGLIKVLTATPTLAM-------------------------------------------------   Hel308 Tga (299) AFHHAGLSRVE-----------RTLIEDAFREGLIKVITATPTLSA-------------------------------------------------   Hel308 Tsi (322) AFHHAGLGRTE-----------RSIIEDAFREGLIKVITATPTLSA-------------------------------------------------   Hel308 Mja (305) AFHHAGLTYQH-----------RKIVEDAFRKRLIKVICCTPTLSA-------------------------------------------------   Consensus  (381) AFHHAGL               R LVEDAFR  LIKVI ATPTLAA                     476                                                                                         570    Hel308 Mbu (342) --------------------------------------GLNLPARRVIIRSYRRFDS-NFG--------------------------MQPIPVLE    Hel308 Afu (335) --------------------------------------GVNLPARRVIVRSLYRFDG-YSK----------------------------RIKVSE    Hel308 Csy (348) --------------------------------------GVNLPARRVVISSVMRYNS-SSGM-------------------------SEPISILE    Hel308 Dth (448) NEAGKCIKSLEKDGELLASRDRSFYYTRARYPHKDVDLRGIGQTYNIFEHSTGEYLGEVDGVRAFKETHPGAVYLHMGETYVVQDLDLETFAVYA    Hel308 Fac (325) --------------------------------------GVNLPARTVIIRDITRFSD---GY-------------------------SKPISGIE    Hel308 Hla (350) --------------------------------------GVNTPARRVIVRDWRRYDG-EFGG-------------------------MKPLDVLE    Hel308 Hpa (344) --------------------------------------GVNTPSRRVIVRDWRRYDG-DIGG-------------------------MQPLDVLE    Hel308 Htu (345) --------------------------------------GVNTPARRVIVRDWRRFDP-SAGG-------------------------MAPLDVLE    Hel308 Hvo (350) --------------------------------------GVNTPSRRVVVRDWQRYDG-DYGG-------------------------MKPLDVLE    Hel308 Mac (342) --------------------------------------GLNLPARRVIIRSYRRYSS-DSG--------------------------MQPIPVLE    Hel308 Mba (342) --------------------------------------GLNLPARRVIIRNYRRYSS-EDG--------------------------MQPIPVLE    Hel308 Mbo (335) --------------------------------------GLNLPARRVIIRDYLRFSA-GEG--------------------------MQPIPVSE    Hel308 Mev (342) --------------------------------------GLNLPARRVIVRNYKRYDP-NFG--------------------------MQPIPVLD    Hel308 Mfe (342) --------------------------------------GLNLPCRRAIVKDLTRYT--NRG--------------------------MRYIPIME    Hel308 Mfr (330) --------------------------------------GLNLPARRVIIRDYLRYSG-GEG--------------------------MRPIPVRE    Hel308 Mhu (335) --------------------------------------GLNLPARRVIIRDYNRFAS-GLG--------------------------MVPIPVGE    Hel308 Mig (343) --------------------------------------GINIPCRRAIVRDLMRFS--NGR--------------------------MKPIPIME    Hel308 Min (324) --------------------------------------GLNLPCRRVIISELKRYT--RRG--------------------------LTYIPIME    Hel308 Mma (339) --------------------------------------GLNLPCRRAIVRDIKRYS--QNG--------------------------LVDIPRME   Hel308 Mmah (342) --------------------------------------GLNLPARRVIIRSYKRYDP-NAG--------------------------MQPIPVLD   Hel308 Mmar (345) --------------------------------------GVNTPSRRVVVRDWRRYDG-SAGG-------------------------MAPLSVLE   Hel308 Mmaz (342) --------------------------------------GLNLPARRVIIRSYRRYSS-DSG--------------------------MQPIPVLE    Hel308 Mok (346) --------------------------------------GINMPCRRAIIRDLKRFS--SRG--------------------------YIPIPKME    Hel308 Mth (337) --------------------------------------GLNLPARRVLIRSYKRYEA-GLG--------------------------TRPIPVME    Hel308 Mzh (342) --------------------------------------GLNLPARRVIIRSYKRYDP-NFG--------------------------MKPIPVLE    Hel308 Nma (345) --------------------------------------GVNTPARRVIVRDWRRFDP-SAGG-------------------------MAPLDVLE    Hel308 Nth (353) --------------------------------------RKLGINQVLQVYSTINDSLVQSLA-------------------------VIDLLLEK    Hel308 Pfu (333) --------------------------------------GINTPAFRVIIRDIWRYS--DFG--------------------------MERIPIIE    Hel308 Sso (351) --------------------------------------GVNLPARTVIIGDIYRFNKKIAGY-------------------------YDEIPIME    Hel308 Tba (355) --------------------------------------GVNLPSFRVIIRDTKRYS--TFG--------------------------WSDIPVLE    Hel308 Tga (334) --------------------------------------GVNLPSFRVIIRDTKRYA--GFG--------------------------WTDIPVLE    Hel308 Tsi (357) --------------------------------------GVNLPAYRVIIRDTKRYS--NFG--------------------------WVDIPVLE    Hel308 Mja (340) --------------------------------------GLNLPCRRAIVKDLTRFTN---KG-------------------------MRYIPIME    Consensus  (476)                                       GLNLPARRVIIRDYKRY     G                          M PIPVLE                     571                                                                                         665    Hel308 Mbu (372) YKQMAGRAGRPHLDPYGESVLLAKTYDEF--AQLMENYVEADAEDIWSKLGTENALRTHVLSTIVNGFASTRQELFDFFGATFFAYQQ-DKWMLE    Hel308 Afu (363) YKQMAGRAGRPGMDERGEAIIIVGKRDR---EIAVKRYIFGEPERITSKLGVETHLRFHSLSIICDGYAKTLEELEDFFADTFFFKQN--EISLS    Hel308 Csy (379) YKQLCGRAGRPQYDKSGEAIVVGGVNAD----EIFDRYIGGEPEPIRSAMVDDRALRIHVLSLVTTSPGIKEDDVTEFFLGTLGGQQS-GESTVK    Hel308 Dth (543) AKSEANYYTRPITEKYTEIVEVQATRATAAGELCLGRLKVTEHVSAYEKRLVRGQARIGLIPLDLPPLVFETQGMWFTLDSQVRRDVEDRRLHFM    Hel308 Fac (354) IQQMIGRAGRPKYDKKGYGYIYAASPG---MLRVAEGYLTGELEPVISRMDSNSLIRFNVLALISSGIATDLKGIQDFYGKTLLAAQN-DIDGYE    Hel308 Hla (381) VHQMCGRAGRPGLDPYGEAVLLANDADTK--EELFERYLWADPEPVRSKLAAEPALRTHVLATVASGFASTRDGLLSFLDNTLYATQTDDEGRLA    Hel308 Hpa (375) VHQMFGRAGRPGLDPHGEAVLIAKSHDEL--QELFDQYVWADPEPVHSKLAAEPALRTHILATVASGFAGTEEELLDFLERTLYATQTDETGRLE    Hel308 Htu (376) VHQMMGRAGRPGLDPYGEAVLLAKSHDES--EELFDRYIWADPEPVRSKLAAEPALRTHVLATIASGFARTRGGLLEFLEATLYASQSSEAGRLE    Hel308 Hvo (381) VHQMMGRAGRPGLDPYGEAVLLAKDADAR--DELFERYIWADAEDVRSKLAAEPALRTHLLATVASGFAHTREGLLEFLDQTLYATQTDDPERLG    Hel308 Mac (372) YKQMAGRAGRPRLDPYGEAVLLAKSYEEL--LFLFEKYIEAGAEDIWSKLGTENALRTHVLSTISNGFARTKEELMDFLEATFFAYQY-SNFGLS    Hel308 Mba (372) YKQMAGRAGRPRLDPYGEAVLVAKSYKEF--VFLFENYIEANAEDIWSKLGTENALRTHVLSTISNGFARTYDELMDFLEATFFAFQY-SNFGLS    Hel308 Mbo (365) YRQMAGRAGRPRLDPYGEAVLIAKEAEQV--PELFEVYIEAEAEDVHSRIAEPTALYTHVLSLVASGFAGTRGELTEFMNRSFYVHEHKQGRLIH    Hel308 Mev (372) YKQMAGRAGRPSLDPYGESVLISHTYNEF--TDLLDRYIDAEPEDILSKLGTENALRTHVLSTIVNGFATTRQGMVDFMGSSFFAYQQ-QKWSLI    Hel308 Mfe (371) IQQCIGRAGRLGLDPYGEGIIVAKNDR---DYLRSYQVLTQKPEPIYSKLSNQAVLRTQLLGLIATIEIRDEYDLEWFIRNTFYAYQYGNLREVA    Hel308 Mfr (360) YRQMAGRAGRPHLDPYGEAILIAKTEYAV--NDLHEEYVEAPDEDVTSRCGEKGVLTAHILSLIATGYARSYDELMAFLEKTLYAYQHTGKKALT    Hel308 Mhu (365) YHQMAGRAGRPHLDPYGEAVLLAKDAPSV--ERLFETFIDAEAERVDSQCVDDASLCAHILSLIATGFAHDQEALSSFMERTFYFFQHPKTRSLP    Hel308 Mig (372) IHQCIGRAGRPGLDPYGEGIIFVKNER---DLERAEQYLEGKPEYIYSKLSNQAVLRTQLLGMIATREIENEFDLISFIKNTFYAHQYGNLGGVL    Hel308 Min (353) VQQCIGRAGRPGLDEYGEGILVAKDER---DYLRALQCLTQKPEPIYSKLSNDSVLRTQILGLIATRYVLDEYDLEEFIKNTFYAYQYKNLDEIK    Hel308 Mma (368) IQQCIGRAGRPGLDPYGEGIIYIKNER---DAEKAYEILTGSVENIYSKLANQKVLRIHILGLISTGEIKDGQNLVNFMKNTFYAHQFGNIGAVL   Hel308 Mmah (372) YKQMAGRAGRPHLDPYGEAVVIVKTYEEF--TDVLERYISASAEDIWSKLGTENALRTHILSTIASGFANCHREILTFLGSTFFAHQQ-QSWNFE   Hel308 Mmar (376) VHQMMGRAGRPGLDPYGEAVLIASSHDEV--DELFERYVWADPEPVRSKLAAEPALRTHILATVASGFARSRKGLLEFLEQTLYASQTDDSGQLE   Hel308 Mmaz (372) YKQMAGRAGRPRLDPYGEAVLLAKSYEEF--VFLFEKYIEAGAEDIWSKLGTENALRTHILSTISNGFARTREELMDFLEATFFAFQY-SNFGLS    Hel308 Mok (375) IHQCIGRAGRPNLDPYGEGIIYINNTENPELIENAKNYLIGNVEEIYSKLSNQKVLRTHMLGLITTGDIKNKNDLEEFIKNTFYAYQYQNTKKIL    Hel308 Mth (367) YRQMAGRAGRPGLDPYGESLIMARSESEL--QKLMDHYVMGEPEDIWSKLASERALRTHVLATIASRFADSVDSLSRLMASTFYARQQ-DPSYLG    Hel308 Mzh (372) YKQMAGRAGRPHLDPYGESVLIARSYDEF--MDIMENYVNADPEDIWSKLGTENALRTHVLSTIVNGFAYTYRGLMDFVKMTFFAYQK-EASDLH    Hel308 Nma (376) VHQMMGRAGRPGLDPYGEAVLLAKSHDES--QELFDRYVWADPEPVRSKLAAEPALRTHVLATIASGFARTREGLLEFLEATLYASQSSEGGRLE    Hel308 Nth (385) WIEPATEYPLPLDILFHQIISICHEANGVRLDPLIDNIKANAAFYKLKEEDINHVINYMIENDFLQLIRNSAELIVGLEGERLLRGKEFYAVFMT    Hel308 Pfu (362) VHQMLGRAGRPKYDEVGEGIIVSTSDD---PREVMNHYIFGKPEKLFSQLSNESNLRSQVLALIATFGYSTVEEILKFISNTFYAYQRKDTYSLE    Hel308 Sso (383) YKQMSGRAGRPGFDQIGESIVVVRDKEDV--DRVFKKYVLSDVEPIESKLGSERAFYTFLLGILSAEGNLSEKQLENFAYESLLAKQL-----VD    Hel308 Tba (384) IQQMIGRAGRPKYDKEGEAIIVAKTEK---PEELMEKYIFGKPEKLFSMLSNDAAFRSQVLALITNFGVESFRELIGFLEKTFYYHQRKDLEILE    Hel308 Tga (363) IQQMMGRAGRPRYDKYGEAIIVARTDE---PGKLMERYIRGKPEKLFSMLANEQAFRSQVLALITNFGIRSFPELVRFLERTFYAHQRKDLSSLE    Hel308 Tsi (386) IQQMMGRAGRPKYDIEGQAIIIAKTEK---PEDLMKRYVLGKPEKLFSMLSNEASFRSQVLALITNFGVGNFKELVNFLERTFYYHQRKNLEALE    Hel308 Mja (369) IQQCIGRAGRPGLDPYGEGIIVAKNDRDY---LRAYQALTQKPEPIYSKLSNQAVLRTQLLGLIATGEIRDEYDLEWFIRNTFYAHQYGNLREVA    Consensus  (571) I QM GRAGRP LDPYGEAVLIAKS D     EL E YI ADPE IWSKLA E ALRTHVLALIASGFA T ELLDFL   TFYAYQ       L                     666                                                                                         760    Hel308 Mbu (464) EVINDCLEFLIDKAMVSET-E--------------------------------------DIEDASKLFLRGTRLGSLVSMLYIDPLSGSKIVDGF    Hel308 Afu (453) YELERVVRQLENWGMVVEAAH-----------------------------------------------LAPTKLGSLVSRLYIDPLTGFIFHDVL    Hel308 Csy (469) FSVAVALRFLQEEGMLGRR----------------------------------------------GGRLAATKMGRLVSRLYMDPMTAVTLRDAV    Hel308 Dth (638) GGLHALEHGLIGCMPLIILTDRNDLGGIASPVHEQLHKG--------------AVFIYDGTPGGIGLCRQAFELGDRLVARAMGILSSCTCENGC    Hel308 Fac (445) LAFESALYFLKDNDFITEEN----------------------------------------------DIYSATKFGRLTSDLYIDPVSSLILKKCL    Hel308 Hla (474) AVTDTVLDYLAVNDFIERDRD------------------------------------------GGSESLTATGIGHTVSRLYLDPMSAAEMIDGL    Hel308 Hpa (468) TVTQHVLDYLDRNGFLERDD-----------------------------------------------RLRATGLGHRVSQLYLDPMSAAEIIDGL    Hel308 Htu (469) SVTDDVLDYLERNDFIERSR--DDEAEDSGEDDGPFTSAADLAEQ---------QAAK------REETLEATSLGHTVSRLYLDPMSAAEIVHGL    Hel308 Hvo (474) QVTDRVLDYLEVNGFVEFEG----------------------------------------------ETIQATPVGHTVSRLYLDPMSAAEIIDGL    Hel308 Mac (464) VVVDECLNFLRQEGMLEQDS----------------------------------------------DALISTMFGKLVSRLYIDPLSAALIAKGL    Hel308 Mba (464) TVVNECLNFLRQEGMLEKD-----------------------------------------------DALIPTSFGKLVSRLYIDPLSAARIAKGL    Hel308 Mbo (458) RAIDEALQFLITAEMVVEV----------------------------------------------GEHIGATELGTLVSRMYIDPRSAFAIVTTL    Hel308 Mev (464) DVVDDCIEFLQDNEMIKD-----------------------------------------DG--ER---LYATRLGQVISTLYIDPLSGAIIIDKL    Hel308 Mfe (463) KNINEVIRFLEEK--------------------------------------------------EFMIDFIPTELGKRVAELYIDPLSAKYMIDGL    Hel308 Mfr (453) RTLDDALGFLTEAEMVTDL----------------------------------------------SGMLHATEYGDLTSRLYIDPHSAEIITTAL    Hel308 Mhu (458) RLVADAIRFLTTAGMVEER----------------------------------------------ENTLSATRLGSLVSRLYLNPCTARLILDSL    Hel308 Mig (464) RNIKEVINFLEEN--------------------------------------------------DFIADYFPTKLGKRVSELYIDPLSAKIIIDGL    Hel308 Min (445) KKIKEIIEFLEDCN--------------------------------------------------FIKNFEVTPLGKKVSNLYLDPLSAKIMIDNI    Hel308 Mma (460) LNVSEVVEFLEKNKFLETTIHKKTENKVRELSFDS------S-NN---LVLDSKETSFDLTNPNSNIEFRSTKLGKRISELYIDPMSSEIIIEEL   Hel308 Mmah (464) ELLEDCLIFLKNEGMLEQD-N--------------------------------------ET-------IRATELGKMISSLYIDPLSASKIIRGL   Hel308 Mmar (469) RVVDDVLTYLQRNDFLEIEAG----------------------------------------------ELDATSLGHTVSRLYLDPMSAAEIVDGL   Hel308 Mmaz (464) AVVDECLDFLRREGMLEKDP----------------------------------------------DALVSTVFGKLVSRLYIDPLSAALIAKGL    Hel308 Mok (470) ENIYEITNFLEKNGFIELNYRRDENKDKSNNSHNNKKNISNTNNSIKMLVLDNNNSLTIKSRHEEDVYYNITPLGKKVSELYIDPLSAEYIIDGL    Hel308 Mth (459) ETIASVLEFLVRSDMIDKD-------------------------------------------------LTPTPLGALVSRLYIDPLSAMVMIQEI    Hel308 Mzh (464) DVIEECVRFLIDNEMIISD-S--------------------------------------NDILPES-AFRSTATGKLISMLYIDPLSGSLIMDGI    Hel308 Nma (469) RVTDDVLSYLERNDFIERSGGPEDTLNSEADAASAFTSAADLADS---------DGGDSGGTTGQEEDLEATSLGHTVSRLYLDPMSAAEIVHGL    Hel308 Nth (480) QEEFEVREGIRKIGSIDKS-------------------------------------------LMVSEGDNIILAGQLWTIKNIDIERDIIYVAKA    Hel308 Pfu (454) EKIRNILYFLLEN---------------------------------------------EFIEISLEDKIRPLSLGIRTAKLYIDPYTAKMFKDKM    Hel308 Sso (471) VYFDRAIRWLLEHSFIKEE----------------------------------------------GNTFALTNFGKRVADLYINPFTADIIRKGL    Hel308 Tba (476) GKAKSIVYFLLEN---------------------------------------------EFIDIDLNDSFIALPFGIRTSQLYLDPLTAKKFKDAL    Hel308 Tga (455) YKAKEVVYFLIEN---------------------------------------------EFIDLDLEDRFIPLPFGKRTSQLYIDPLTAKKFKDAF    Hel308 Tsi (478) GKAKSIVYFLFEN---------------------------------------------EFIDIDLNDQFMPLPLGIRTSQLYLDPVTAKKFKDAF    Hel308 Mja (461) KNINEVIRFLEENEFI--------------------------------------------------IDFMPTELGKRVSELYIDPLSAKFIIDGL    Consensus  (666)   I EVL FL  N  I                                                    L AT LG  VS LYIDPLSA  IIDGL                     761                                                                                         855    Hel308 Mbu (520) KDIGKSTGGNMGSLEDDKG----------------------------------------------------DDITVTDMTLLHLVCSTPDMRQLY    Hel308 Afu (501) SRMELS-----------------------------------------------------------------------DIGALHLICRTPDMERLT    Hel308 Csy (518) GEASPGR--------------------------------------------------------------------MHTLGFLHLVSECSEFMPRF    Hel308 Dth (719) PGCIHSPKCGSGNR------------------------------------------------------------PLDKEAAMHMLAVLAGERCGE    Hel308 Fac (494) DLEFS-------------------------------------------------------------------------ELYLYYISKTPDMLTFN    Hel308 Hla (527) RSVARDAADTGASAEADNG-EFVRTGDADDASGGDEPGFGTYTRAGDDESGER-------TENEETDEEETEASEVTPLGLYHLISRTPDMYELY    Hel308 Hpa (516) RDADG---------------------------------------------------------------------KPTALGLYHLVSRTPDMYQLY    Hel308 Htu (547) ERADER---------------------------------------------------------------------PTALGLYQLVSRTPDMYELY    Hel308 Hvo (523) EWAADHRTEKLRALAGETPEKPTRDRSESDESGGFQRASEMVADDGDGGGGEDGVGANGDGDSDDADGVETDRTYPTPLGLYHLVCRTPDMYQLY    Hel308 Mac (513) REAGT----------------------------------------------------------------------LTELTLLHLVCSTPDMRLMY    Hel308 Mba (512) KGAKS----------------------------------------------------------------------LSELTLLHLVCSTPDMRLLY    Hel308 Mbo (507) REQEK----------------------------------------------------------------------YADLGLIQLICTTPDMPTLY    Hel308 Mev (513) KKADK-----------------------------------------------------------------------TDMTMLHIICSTPDMRQLY    Hel308 Mfe (508) NEMENED----------------------------------------------------------------------DIYYLYLISKTLEMMPNL    Hel308 Mfr (502) REEGE-----------------------------------------------------------------------TDLALLQLLCMTPDMFTLY    Hel308 Mhu (507) KSCKT----------------------------------------------------------------------PTLIGLLHVICVSPDMQRLY    Hel308 Mig (509) KEMGNVDNE--------------------------------------------------------------------ELYYLYLISKTLEMMPLL    Hel308 Min (490) EVKDDLH-------------------------------------------------------------------------LLYILCKCIEMKPLL    Hel308 Mma (545) HELKKKCDQLDR--------------------------------------------------------------SKIDQYLFYLISKTNEMRPLL   Hel308 Mmah (513) EKTTH----------------------------------------------------------------------VTDMTLLQLICSTPDMRLLY   Hel308 Mmar (518) RDWERGASDSTSASGSPAD----AQAEP-PANSGFTTASELAEDADESDADRD--------------------DDISALGLYHLVSRTPDMYQLY   Hel308 Mmaz (513) REAGT----------------------------------------------------------------------LTELTLLHLICSTPDMRLMY    Hel308 Mok (565) KNLHKKTLSNPKNM------------------------------------------------------------ECYILHILYIISKTTEMQPVL    Hel308 Mth (505) RGIRR----------------------------------------------------------------------PTVLTLLHVITMTPDMELLF    Hel308 Mzh (519) RKADY----------------------------------------------------------------------FEDITMMHLICSTPDMKNLY    Hel308 Nma (555) EDADER---------------------------------------------------------------------PTALGLYQLVSRTPDMYELY    Hel308 Nth (532) VDGKPPK--------------------------------------------------------------------YSGGGFILNPKIPERMHKIL    Hel308 Pfu (504) EEVVKDPN---------------------------------------------------------------------PIGIFHLISLTPDITPFN    Hel308 Sso (520) EGHKAS----------------------------------------------------------------------CELAYLHLLAFTPDGPLVS    Hel308 Tba (526) PQIEENPN---------------------------------------------------------------------PLGIFQLLASTPDMGTLS    Hel308 Tga (505) PAIERNPN---------------------------------------------------------------------PFGIFQLIASTPDMATLT    Hel308 Tsi (528) EKLEKNPN---------------------------------------------------------------------PLGIFQLLASTPDMSSLR    Hel308 Mja (506) EEMENEE----------------------------------------------------------------------EIYYLYLISKTLEMMPNL    Consensus  (761)                                                                               LGLLHLIS TPDM  LY                     856                                                                                         950    Hel308 Mbu (563) LRNTDYTIVNEYIVAHSDEFH---EIPDKLKETDYEWFMGEVKTAMLLEEW---------------VTEVSAEDITRHFNVGEGDIHALADTSEW    Hel308 Afu (525) VRKTDSWVEEEAFRLRKELSY----YPSDFS-VEYDWFLSEVKTALCLKDW---------------IEEKDEDEICAKYGIAPGDLRRIVETAEW    Hel308 Csy (545) ALRQKDHEVAEMMLEAGRGELLR---P--------VYSYECGRGLLALHRW---------------IGESPEAKLAEDLKFESGDVHRMVESSGW    Hel308 Dth (754) AKRKDVSCRIETDEGSMEIDSG-YTKSDQAELPYAVLDIETRYSAQEVGGWGNCHRMGVSFAVVFDSRNQEFVTFDQEQAADLGSFLEDFSLVVG    Hel308 Fac (517) YRASDYEYLEEFLDRHNISDFS-------------EESMGAAKTAIILNEW---------------INEVPINTIAETFGIGPGDIQAKASSADW    Hel308 Hla (615) LKSGDRETYTELCYERETEFLG--DVPSEYEDVRFEDWLASLKTARLLEDW---------------VNEVDEDRITERYGVGPGDIRGKVDTAEW    Hel308 Hpa (542) LRSGDRERYTEIAYEREPEFLG--HMPSEFEDNAFEDWLSALKTARLLEDW---------------ASELDEDRITERYAIGPGDIRGKVETAQW    Hel308 Htu (573) LRSGEDEKFGELFYERETELLG--DAPSEYEEDRFEDWLAALKTGKLLEDW---------------ADETDEETITDRYKIGPGDLRGKVDTAEW    Hel308 Hvo (618) LKSGDRETYTELCYEREPEFLG--RVPSEYEDVAFEDWLSALKTAKLLEDW---------------VGEVDEDRITERYGVGPGDIRGKVETSEW    Hel308 Mac (538) MRSQDYQDINDFVMAHAEEFS---KVPSPFNIVEYEWFLSEVKTSLLLMDW---------------IHEKPENEICLKFGTGEGDIHTTADIAEW    Hel308 Mba (537) MRSHDYQDINDYVMAHASEFV---KVPSPFDTTEYEWFLGEVKTSLLLLDW---------------IHEKSENEICLKFGTGEGDIHSIADIAEW    Hel308 Mbo (532) AKNADLPALSRMLEVRGADIW---LPP-PLDDDAAETYYRAVKTAMLLSDW---------------TDELSEEKICERYGVGPGDVFGMVENINW    Hel308 Mev (538) LRSKEYEKINEYVMTHSDEFV---EVPNPFKSIEYEWFLGEVKTALLINEW---------------IDEKTLDDITAEFGVGEGDINALSDISEW    Hel308 Mfe (533) RVYKSEE--LNLIDEMENLG------IKSFE----IEDLEAFKTAKMLYDW---------------ISEVPEDEILKKYKIEPGILRYKVENAVW    Hel308 Mfr (527) VKKNDLGTLEKFFFEHEEEFR---T---EFSYDEMEDFFRSLKTAMLLSDW---------------TDEIGDDTICTRFGVGPGDIFNAVQGISW    Hel308 Mhu (532) LKAADTQLLRTFLFKHKDDLI---LPL-PFEQEEEELWLSGLKTALVLTDW---------------ADEFSEGMIEERYGIGAGDLYNIVDSGKW    Hel308 Mig (536) RVNSFEE--LDLILEMEEAG------IYDRT----YDDLAAFKNAKMLYDW---------------INEVPEDEILKKYKIEPGILRYKVEQAKW    Hel308 Min (512) RVYRKEE--EELAEELLNYE------I-FIS----YENLEEFKTAKMLYDW---------------INEVPEDEILKTYKVEPGILRYKVEVAKW    Hel308 Mma (578) RIRPNEE--LDLILEMDKMG------LKDYS----IENIEAFKNSKMFCDW---------------VSEIPEEIILEKYGVEPGILRYKVEQAKW   Hel308 Mmah (538) LRNRDYEIINDYVMNHTEEFI---EVPSPFKQIEYEWFLSEVKTALLLLEW---------------INEKSLEKIVENYQVGEGDIYASSDIAEW   Hel308 Mmar (589) LRSGDREEYEMELFEREEELLG--PTPSEFEEGRFEDWLSALKTARLLEDW---------------ATEVDEATITDRYGVGPGDIRGKVETAQW   Hel308 Mmaz (538) MRSQDYQEVNDYVMAHAGEFS---KVPNPFNIAEYEWFLGEVKTSLLLMDW---------------IHEKPENEICLKFGIGEGDIHATADIAEW    Hel308 Mok (600) RVRRKEE--NDLINDMIKLDIDVDDVIYGIS----SENLEYFKNAKLFYDW---------------INEIPEEELLLGYNIEPGILRYNVEQAKW    Hel308 Mth (530) VQQS-DNWLEDFISEHSSELG---NEKN------FDWLLREVKTASMLMDW---------------INEVHEDRIEDRYSISPGDLVRIAETAEW    Hel308 Mzh (544) MRSSDYENVNMYVLQNKDKFI---SMPSPFKMIEYEWFLGEVKTALLLLDW---------------INEVPADDICKKYGIGEGDIRMFSETAVW    Hel308 Nma (581) LRSGEDEKFGELYYERERELLG--DAPSEFEEERFEDWLAALKTGKLLEDW---------------ATEDDEEQITERYKIGPGDLRGKVDTAEW    Hel308 Nth (559) CERKNFEFIDNMAQNHLEEQR---------------------KPFELYNIK---------------PNERVIWNNGDEILFETYTGTKIFQTLAW    Hel308 Pfu (530) YSKREFERLEEEYYEFKDRLYFDDPYISGYDPYLERKFFRAFKTALVLLAW---------------INEVPEGEIVEKYSVEPGDIYRIVETAEW    Hel308 Sso (545) VGRNEEEELIELLEDLDCELL----IEEPYEEDEYSLYINALKVALIMKDW---------------MDEVDEDTILSKYNIGSGDLRNMVETMDW    Hel308 Tba (552) IKRKEQESYLDYAYEMEDYLYRSIPYWEDYE---FQKFLSEVKTAKLLLDW---------------INEVSEAKLIEAYGIDTGDLYRIIELADW    Hel308 Tga (531) ARRREMEDYLDLAYELEDKLYASIPYYEDSR---FQGFLGQVKTAKVLLDW---------------INEVPEARIYETYSIDPGDLYRLLELADW    Hel308 Tsi (554) VKRKEQEDLLDYAYEMEEYLYQNIPYWEDYK---FEKFLGETKTAKLLLDW---------------INEVNDVKILETYEIDTGDLYRILELVDW    Hel308 Mja (531) RVYNSEE--LNLIDEMDSLGIK----------SFEIEDLEAFKTAKMLYDW---------------INEVPEDEILKRYKIEPGILRYKVENAVW    Consensus  (856) LR  D E L E I E   E          F    FE FL  VKTA LL DW               I EV ED I ERYGIGPGDL   VE AEW                     951                                                                                        1045   Hel308 Mbu (640) LMHAAAKLAELLGVEYSS--------HAYSLEKRIRYGSGLDLMELVGIRGVGRVRARKLYNAGFVS---------VAKLKGADISVLSKLVGP-    Hel308 Afu (600) LSNAMNRIAEEVG-N--T--------SVSGLTERIKHGVKEELLELVRIRHIGRVRARKLYNAGIRN---------AEDIVRHREKVASLIGRG-    Hel308 Csy (614) LLRCIWEISKHQERPDLLG-------ELDVLRSRVAYGIKAELVPLVSIKGIGRVRSRRLFRGGIKG---------PGDLAAVPVERLSRVEGIG    Hel308 Dth (848) FNLLKFDYRVLQGLSDYDFSSLPTLDMLREIEARLGHRLSLDHLARHTLGTNKSANGLMALKWWKEGELDKIVEYCRQDVSVTRDLYLFGRDKGY    Hel308 Fac (584) ISYSLYRLGSMFDKENEN--------NLLHLNIRIKEGVKEEIIRIIEIPQVGRVRGRRLYNNGFKS---------IDDIANARVEDISRIFGFS    Hel308 Hla (693) LLRAAETLARDVEGVDGDVVV-----AVREARKRIEYGVREELLDLAGVRNVGRKRARRLFEAGIET---------RADLREADKAVVLGALRGR    Hel308 Hpa (620) LLNAAERLAAELQRDDAEGIPSATTTAVREARKRVEYGVEEELLDLAGVRNVGRKRARRLYEAGIES---------RADLREADKSVVLGALRGR    Hel308 Htu (651) LLGAAESLAAEIDSEWTV--------AVREARARVEHGVGEELLELVSVGGVGRKRARRLYDAGIEE---------PADLRSADKGIVLSVLKG-    Hel308 Hvo (696) LLGAAERLATELD---LDSVY-----AVREAKKRVEYGVREELLDLAGVRGVGRKRARRLFEAGVET---------RADLREADKPRVLAALRGR    Hel308 Mac (615) IMHVATQLARLLDLKGAK--------EAAELEKRIHYGAGPELMDLLDIRGIGRVRARKLYGAGFKS---------TADLAGATPEKVAALVGP-    Hel308 Mba (614) IMHVTSQLAGLLDLKGAR--------EAAELEKRIHYGAAPELIDLLNIRGIGRVRARKLYEAGFKS---------SAELAEVDPEKVAALLGP-    Hel308 Mbo (608) LLHATSQLARMFVPKFYG--------QIADCEICMKNGIRRELLPLVRLRGIGRVRARRLFNNGITS---------PEELSRHKKEDLVKILGS-    Hel308 Mev (615) LMHSAVNLANLTDLDAD---------KAQELEKRIHHGVNKDLIQLVSISNIGRVRARKLYEAGIQS---------VSDIKNTKLHILSNYLGR-    Hel308 Mfe (601) LMHALKEMAKIIGKN---------SEIPEKLEIRLEYGAKEDIIELLNVKYIGRVRARKLYNAGIRN---------VEDIINNPSK---VASIIG    Hel308 Mfr (601) LLHASGRLARLVAPEHRD--------AVEETTLRVRHGIRRELIPLVRVKGIGRVRARRLFNNGITG---------PELLAAADPSVVGHIVGG-    Hel308 Mhu (608) LLHGTERLVSVEMPEMSQ--------VVKTLSVRVHHGVKSELLPLVALRNIGRVRARTLYNAGYPD---------PEAVARAGLSTIARIIGE-    Hel308 Mig (604) MIYSTKEIAKLLNRN---------IDTLSKLEIRLEYGAKEDIIELLKIKYVGRARARKLYDAGIRS---------VEDIINNPKK---VASLLG    Hel308 Min (579) LSYSLKEIAKILNKEVP------------NLELRLEYGAKEELLELLKIKYIGRVRARKLYSAGIRN---------REDIIKNPKK---VANILG    Hel308 Mma (646) MIYSTKEIAKLIHLDNSE-----IYKSLLKMEVRIEYGAKEELIELLNVKNVGRIRSRKLYDAGIRS---------KIEINKNPEK---ILELFG   Hel308 Mmah (615) LMHATQRIASRINPQLET--------ECAKLEKRIHYGAGSELIELVEIPNVGRARARKLFKKGYRS---------RQKLATADEKQLAGIVGP-   Hel308 Mmar (667) LLGAAESLASEVDLDAAR--------AISEARIRVEHGVREELVDLAGVRGVGRKRARRLFQAGITD---------RAQLRDADKAVVLAALRGR   Hel308 Mmaz (615) IMHVTAQLAGLLDLKGAK--------EASELEKRIRYGAAPELMDLLDIRSVGRVRARKLYEAGFKS---------TAELAAASPEHIAVLVGP-    Hel308 Mok (674) MIHSAKEIFNLLNIDNKV-----IKDCLNDLEIRMEYGAKQDIIELLKIKHIGRARARILYNAGIKN---------ANDIINNQKN---IINLLG    Hel308 Mth (600) LMSALHRISKHMDLGVTY--------LAERLALRIHYGAGDELLQLLELKGIGRVRARKLYQAGYRS---------LEDLKAADKSTLSEILGP-    Hel308 Mzh (621) LMHATSRLSGLLKVSEASE-------KSKELEKRLSYGINSELVNIVALKGIGRVRARKIYENGYRS---------IDDLKKADPLKLSKIVGS-    Hel308 Nma (659) LLGAAESLASEIDSEWAV--------AVREARARVEHGVGEELLELVSVSGIGRKRARRLYAAGIEE---------PAALRSADKGVILHVLKG-    Hel308 Nth (618) ILRSYNVNIKEIDGIGRIN-----IEGGIDLPGVLQDIKETDWRPEYLLDFTLEQEKFKSKFSPYLP---------KDLQDKMHIAHLVDIEGVK    Hel308 Pfu (610) LVYSLKEIAKVLG-AYE------IVDYLETLRVRVKYGIREELIPLMQLPLVGRRRARALYNSGFRS---------IEDISQARPEELLKIEGIG    Hel308 Sso (621) LTYSAYHLSRELKLNEHAD-------KLRILNLRVRDGIKEELLELVQISGVGRKRARLLYNNGIKE---------LGDVVMNPDKVKNLLGQK-    Hel308 Tba (629) LMYSLIELAKVLNAGGE------TIKYLRRLHLRLKHGVREELLELVELPMIGRRRARALYNAGFKN---------VNDIVKAKPSELLAVEGIG    Hel308 Tga (608) LMYSLIELYKLFEPKEE------ILNYLRDLHLRLRHGVREELLELVRLPNIGRKRARALYNAGFRS---------VEAIANAKPAELLAVEGIG    Hel308 Tsi (631) LMYSLIELYKLFDPKPE------VLDFLKKLHIRVKHGVREELLELITLPMIGRKRARALYNAGFKG---------IDDIVRAKASELLKVEGIG    Hel308 Mja (599) IMHALKEIAKLIGKSSDI---------PEKLEIRLEYGAKEDIIELLSIKYIGRVRARKLYNAGIRS---------IEDIINNPSK---VASIIG    Consensus  (951) LMHA   LAKLL               L EL IRI YGVKEELLELV IR IGRVRARKLY AGIRS           DL  A    L  ILG                     1046                                                                                       1140    Hel308 Mbu (717) -KVAYNILSGIGVRVNDKHFNSAPISSNTLD--------------------TLLDKNQKTFNDFQ------------------------------    Hel308 Afu (674) --IAERVVEGISVKSLNPESAAALEHHHHHH----------------------------------------------------------------   Hel308 Csy (693) ATLANNIKSQLRKGG--------------------------------------------------------------------------------   Hel308 Dth (943) LLFKNKAGKKVRIPVSWQDTAFQV-----------------------------------------------------------------------   Hel308 Fac (662) TKLAKDIIENAGKLNNRYYR---------------------------------------------------------------------------   Hel308 Hla (774) ERTAERILEHAGREDPSMDDVRPDKSASAAATAGS--------------ASDEDGEGQASLGDFR------------------------------    Hel308 Hpa (706) KKTAENILENVGRQDPSLDDVEADAET---------AA---------TSARATNDGGQQSLGDFE------------------------------    Hel308 Htu (728) EKTAENILENAGREDPSMDGVEPADGGPAVGAATNGSSGGSETDETGRADAAESDDSQSSLGDF-------------------------------    Hel308 Hvo (774) RKTAENILEAAGRKDPSMDAVDEDDAPDDAVPDDA---G--------FETAKERADQQASLGDFEGS----------------------------    Hel308 Mac (692) -KIAERIFRQIGRREAVSEISDSERLEKS------------------------SQDGQSTISDF-------------------------------   Hel308 Mba (691) -KIADRIFKQIRGRGTSSGIIASEPPEKS------------------------PYSGQKTISDY-------------------------------    Hel308 Mbo (685) -GIAEQVLEQLHPSKDTGKKEPPSGDKNTN------------------------PG-QSTLFHFG------------------------------    Hel308 Mev (691) -KTAYKVLEQLGVEPEDNQQIDEEPESIKSY--------------------SGNDQGQKTFNDF-------------------------------   Hel308 Mfe (675) EKITKKILEDLG--IKFGQ---------------------------------------QKLIF--------------------------------   Hel308 Mfr (678) -KTAESII---------------------------------------------------------------------------------------   Hel308 Mhu (685) -GIARQVIDEITGVKRSGIHSSDDDYQQKT------------------------PE-LLTDIPGIGKKMAEKLQNAGIITVSDLLTADEVLLSDV    Hel308 Mig (678) EKIAKKILGELG--MKFGQ---------------------------------------QTLQI--------------------------------    Hel308 Min (650) EKISKKIFEELG--VRYGQ---------------------------------------QRLI---------------------------------   Hel308 Mma (724) EKIGKKILGEHG--MKYGQ---------------------------------------QTLLNFN------------------------------  Hel308 Mmah (692) -KIAQKILSYLGRETDSNGYVEPETLENK-------------------------KQ-QKTFQDFI------------------------------  Hel308 Mmar (745) RKTAENVLENAGHRDPSMEGVEPAPDVSVDLNDGADGD---------ASAESTANDDQASLGDF-------------------------------   Hel308 Mmaz (692) -KITERIFKQIGRREAVSEFSDIEPLEKG------------------------SSDGQRTISDY-------------------------------    Hel308 Mok (752) EKIARKILSELGVDTKFGQ---------------------------------------MRLSI--------------------------------    Hel308 Mth (677) -KIAEGVISQLK-EPGVSA----------------------------------------------------------------------------   Hel308 Mzh (699) -KISQKILKQLDIDVDISEIKEKDSDTVP-E--------------------P--ESSQKTISDFT------------------------------    Hel308 Nma (736) EKTAENILENAGREEPSMDGVEPIPVEGGSGSGSSNSSGSSEPNADANATEDDADDNQSSLGDF-------------------------------    Hel308 Nth (699) TFLENKKIKEIKL----------------------------------------------------------------------------------   Hel308 Pfu (689) VKTVEAIFKFLGKNVKISE-----------------------------------KPRKSTLDYFLKS----------------------------   Hel308 Sso (699) -LGEKVVQEAARLLNRFH-----------------------------------------------------------------------------   Hel308 Tba (709) VKVLERIYRHFGVELPLLKNIKDPDKPEDKPKEKP-------------------KPKKGTLDYFLK-----------------------------   Hel308 Tga (688) AKILDGIYRHLGIEKRVTE-----EK----------------------------PKRKGTLEDFLR-----------------------------   Hel308 Tsi (711) IGVIEKIYQHFGVELPTNE-----KK--------K-------------------KVKKGTLDEFFK-----------------------------    Hel308 Mja (673) EKIAKKILDELGVKFGQQKLSFSGGSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK----KL----------------------------------   Consensus (1046)  KIAEKIL  LG                                               TL  F                     1141                                      1186    Hel308 Mbu (761) ----------------------------------------------   Hel308 Afu (703) ----------------------------------------------   Hel308 Csy (708) ----------------------------------------------   Hel308 Dth (967) ----------------------------------------------   Hel308 Fac (682) ----------------------------------------------   Hel308 Hla (825) ----------------------------------------------   Hel308 Hpa (753) ----------------------------------------------   Hel308 Htu (792) ----------------------------------------------   Hel308 Hvo (830) ----------------------------------------------   Hel308 Mac (731) ----------------------------------------------   Hel308 Mba (730) ----------------------------------------------   Hel308 Mbo (724) ----------------------------------------------   Hel308 Mev (734) ----------------------------------------------   Hel308 Mfe (697) ----------------------------------------------   Hel308 Mfr (685) ----------------------------------------------   Hel308 Mhu (754) LGAARARKVLAFLSNSEKENSSSDKTEEIPDTQKIRGQSSWEDFGC    Hel308 Mig (700) ----------------------------------------------   Hel308 Min (671) ----------------------------------------------   Hel308 Mma (748) ----------------------------------------------  Hel308 Mmah (730) ----------------------------------------------  Hel308 Mmar (800) ----------------------------------------------  Hel308 Mmaz (731) ----------------------------------------------   Hel308 Mok (776) ----------------------------------------------   Hel308 Mth (694) ----------------------------------------------   Hel308 Mzh (740) ----------------------------------------------   Hel308 Nma (800) ----------------------------------------------   Hel308 Nth (712) ----------------------------------------------   Hel308 Pfu (721) ----------------------------------------------   Hel308 Sso (716) ----------------------------------------------   Hel308 Tba (756) ----------------------------------------------   Hel308 Tga (721) ----------------------------------------------   Hel308 Tsi (745) ----------------------------------------------   Hel308 Mja (730) ----------------------------------------------   Consensus (1141) ---------------------------------------------- Thenumber below the * indicates the SEQ ID NO. The “-” are shown foralignment purposes only and do not form part of the sequences.

The invention claimed is:
 1. A method of characterising a targetpolynucleotide, comprising: (a) contacting the target polynucleotidewith a transmembrane pore under an applied voltage field such that aportion of the target polynucleotide is captured by the transmembranepore, wherein the pore is present in a membrane; (b) contacting thetarget polynucleotide with a Hel308 helicase on the cis side of themembrane such that the helicase binds to the target polynucleotide,thereby controlling the movement of the target polynucleotide throughthe transmembrane pore against the applied voltage field from the transside of the membrane to the cis side of the membrane; and (c) measuringone or more characteristics of the target polynucleotide as the targetpolynucleotide moves through the transmembrane pore, therebycharacterising the target polynucleotide.
 2. The method according toclaim 1, wherein the one or more characteristics are selected from (i)the length of the target polynucleotide, (ii) the identity of the targetpolynucleotide, (iii) the sequence of the target polynucleotide, (iv)the secondary structure of the target polynucleotide and (v) whether ornot the target polynucleotide is modified.
 3. The method according toclaim 2, wherein the target polynucleotide is modified by methylation,by oxidation, by damage, with one or more proteins, or with one or morelabels, tags, or spacers.
 4. The method according to claim 1, wherein atleast a portion of the target polynucleotide is double stranded.
 5. Themethod according to claim 1, wherein the transmembrane pore is a proteinpore.
 6. The method according to claim 5, wherein the transmembraneprotein pore is selected from the group consisting of:.alpha.-hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA),outer membrane porin F (OmpF), outer membrane porin G (OmpG), outermembrane phospholipase A, Neisseria autotransporter lipoprotein (NalP)and WZA.
 7. The method according to claim 1, wherein the Hel308 helicasecomprises the amino acid motif Q-X₁-X₂-G-R-A-G-R (SEQ ID NO: 8), whereinX₁ is C, M or L and X₂ is any amino acid residue.
 8. The methodaccording to claim 7, wherein X₂ is A, F, M, C, V, L, I, S, T or P. 9.The method according to claim 1, wherein the Hel308 helicase binds tothe target polynucleotide at an internal nucleotide.
 10. The methodaccording to claim 1, wherein the one or more characteristics of thetarget polynucleotide are measured by electrical measurement and/oroptical measurement.
 11. The method according to claim 10, wherein theelectrical measurement is a current measurement, an impedancemeasurement, a tunnelling measurement, or a field effect transistor(FET) measurement.
 12. The method according to claim 1, wherein at leasta portion of the polynucleotide is double stranded.
 13. The methodaccording to claim 1, wherein, prior to the movement of the targetpolynucleotide through the transmembrane pore from the trans side of themembrane to the cis side of the membrane, the target polynucleotidemoves from the cis side of the membrane to the trans side of themembrane.
 14. The method according to claim 13, wherein the portion ofthe target polynucleotide that is captured by a transmembrane pore isthe 3′ end of the target polynucleotide.
 15. The method according toclaim 1, wherein the portion of the target polynucleotide that iscaptured by a transmembrane pore is the 5′ end of the targetpolynucleotide.
 16. The method according to claim 1, wherein the targetpolynucleotide is partially translocated through the pore in (a). 17.The method according to claim 1, wherein method is carried out using asalt concentration of at least 0.3 M or at least 1.0 M and the salt isoptionally KCl.
 18. The method according to claim 1, wherein an initialhelicase controls the movement of the target polynucleotide into thepore in (a).
 19. The method according to claim 18, wherein the Hel308helicase of (a) binds to an internal polynucleotide of the targetpolynucleotide at a location on the polynucleotide different than theinitial helicase.
 20. The method according to claim 1, wherein thetransmembrane pore is a solid state pore.